Treatment of immune disease by mucosal delivery of antigents using genetically modified lactobacillus

ABSTRACT

The present invention relates to the treatment of autoimmune and allergic diseases by mucosal delivery by micro-organism, in particular  Lactococcus lactis , of secreted immunodominant antigens.

The present invention relates to the treatment of autoimmune andallergic diseases by mucosal delivery by micro-organisms, in particularLactococcus lactis, of secreted immunodominant antigens.

FIELD OF THE INVENTION

The immune system has the task of distinguishing between self andnon-self. The mucosal immune system, present along the respiratory,gastrointestinal and genitourinary tracts, has the additional burden ofcoexisting with an abundance of bacteria and innocuous antigens, such asfood, airborne antigens or the commensally bacterial flora. A keyfeature of the mucosal immune system is its ability to remain tolerantto these antigens while retaining the capacity to repel pathogenseffectively. Introduction of antigen systemically, whether by injectionor injury, leads to local infiltration of inflammatory cells andspecific immunoglobulin production. By contrast, antigens introduced atmucosal surfaces, such as the gastrointestinal and genitourinary tracts,elicit active inhibition of the immune response to those antigenssystemically. The specific induction of these regulated responses byadministration of antigen through the gastrointestinal tract is known asoral tolerance. Oral administration of antigen can lead to systemicunresponsiveness and is an attractive alternative to immunosuppressivemedical inventions that have undesirable side-effects (such assteroids). The invention lies in particular in the field of low-dosetolerance, obtained by continued exposure to low doses of antigen.Tolerance inductions via the mucosa have been proposed as a treatmentstrategy against autoimmune, allergic and inflammatory diseases.

STATE OF THE ART

The following discussion of the background of the invention is merelyprovided to aid the reader in understanding the invention and is notadmitted to describe or constitute prior art to the present invention.

Autoimmune, allergic and inflammatory diseases place a tremendous burdenon the patient and society, resulting in decreased quality of life andhuge costs. Moreover, no adequate treatment exists without acceptableside effects or which is socially appropriate. Current treatments forautoimmune disease are largely palliative, generally immunosuppressive,or anti-inflammatory. Non-immune therapies, such as hormone replacementin Hashimoto's thyroiditis or DM Type 1 treat outcomes of theautoaggressive response. Steroidal or NSAID treatment limitsinflammatory symptoms of many diseases. IVIG is used for CIDP and GBS.More specific immunomodulatory therapies, such as the TNFα antagonistsetanercept, have been shown to be useful in treating RA. Nevertheless,these immunotherapies may be associated with increased risk of adverseeffects, such as increased susceptibility to infection. Celiac disease,which can be characterized by chronic small intestinal inflammation, canonly be effectively treated by a socially restrictive diet that requireslifelong abstinence from foods that contain wheat, rye or barley. Whilea strict gluten free diet can lead to healing of the intestine theintolerance to gluten is permanent.

Celiac disease, also known as celiac sprue or gluten-sensitiveenteropathy, is a chronic inflammatory disease that develops from animmune response to specific dietary grains that contain gluten.Diagnosis can be made based on the classical presentation of diarrhoea,fatty stools, abdominal bloating and cramping, weight loss, metabolicbone diseases, anaemia as well as the presence of serum antibodies withspecificity for gliadin and tissue transglutaminase (tTG) (also termedanti-endomysial). The mucosal lesion is localized in the proximal partof the small intestine, and is characterized by villous atrophy, cryptcell hyperplasia, and lymphocytic infiltration of the epithelium andlamina propria, which release proinflammatory cytokines, such as IL-2and IFN-γ, in response to gliadin. Celiac disease may be considered themost common food-sensitive enteropathy in humans, and may appear at anytime in a person's life. The prevalence is in the range of 1:100 to1:300 in Western, Arabian and Indian populations. Apart from gluten, thedisease can be triggered for the first time after surgery, viralinfection, severe emotional stress, pregnancy or childbirth.

Hence, induction of antigen specific oral tolerance would be anattractive therapeutic approach Although oral tolerance was firstdescribed in 1911, it was not until the later 1970s that investigatorsstarted to address the mechanisms involved (Mayer and Shao, 2004a).Several mechanisms have been proposed for the development of oraltolerance, ranging from the deletion of anti-specific T-cells, overimmune deviation and induction of anergy to suppression by Tregs (Mucidaet al., 2005). Most investigators agree that there are two distinct waysof obtaining oral tolerance, the high-dose tolerance, obtained after asingle high dose of antigen, which is based on anergy and/or deletion(Friedman and Weiner, 1994), and the low-dose tolerance, obtained byrepeated exposure to low doses of antigen, mediated by activesuppression of immune responses by CD4⁺ T-cells, including Foxp3⁺, IL-10and/or TGF-β producing regulatory T-cells. Importantly, regulatory Tcells induced through mucosal tolerance have been shown to mediatebystander suppression, a process through which regulatory cells specificfor one protein suppress the response of nearby effector cells toanother protein. Bystander suppression is a further important feature ofantigen-induced suppression because the pool of antigens that induceorgan-specific autoimmunity are largely unknown, and it overrides thephenomenon of epitope spreading. Epitope spreading is a complication ofautoimmune and allergic diseases whereby the initiating immune responseexpands with time to include responses to other antigens.

Targeted and more efficient delivery of molecules for therapeutic andprophylactic applications is a priority for the pharmaceutical industry.Effective strategies should reduce the required dose, increase safetyand improve efficacy by focusing molecules at the desired site ofaction. Mucosal routes of drug and vaccine delivery offer a number oflogistical and biological advantages compared with injection. Oraldelivery is particularly attractive as a result of the ease ofadministration. However, gastrointestinal degradation and low levels ofabsorption generally render this route of peptide and protein drugdelivery ineffective. Alternative mucosal routes such as the nasal,rectal, pulmonary and ocular routes are also being investigated.

Thus, there remains a problem in the art to effectively induce toleranceof antigens.

SUMMARY OF THE INVENTION

Surprisingly, we found that an immunodominant antigen which isdelivered, and preferably continuously present, at a mucosal site of apatient induces an antigen-specific immunotolerance. In particular, whena micro-organism such as preferably Lactococcus lactis (LL), whichconstitutively expresses and secretes an immunodominant antigen, isdelivered daily at a mucosal site, an antigen-specific immune tolerancewas induced. We observed that the mucosal delivery of such an antigen bya L. lactis micro-organism gives a significantly better suppression ofthe antigen-specific immune response in comparison to the sole mucosaldelivery of said antigen or said micro-organism.

We demonstrate that the invention can induce oral tolerance with muchmore higher efficiency than with monotherapy with antigen or control L.lactis alone. In vivo activation of antigen-specific regulatory T cellswas strongly enhanced. Specifically, mucosal delivery of a gliadinderived peptide, which is immunodominant for DQ8 mediated T-cellresponses by genetically modified L. lactis, induces suppression oflocal and systemic DQ8 restricted T-cell responses. Treatment resultedin an antigen-specific decrease of the proliferative capacity of thesplenocytes and inguinal lymph node cells, which was criticallydependent on the production of IL-10 and TGF-β and associated with asignificant induction of Foxp3+ regulatory T-cells. Because thisapproach of antigen-delivering bacteria has the capacity forpotentiating oral tolerance even in the setting of establishedhypersensitivity, it is applicable for the treatment of celiac diseaseand other autoimmune and/or allergic diseases. The efficacy of theinvention was demonstrated in autoimmune and allergic disease mousemodels, as well as in the context of immune inactivation oftherapeutics.

DETAILED DESCRIPTION OF THE INVENTION

Throughout this disclosure, various publications, patents and publishedpatent specifications are referenced by an identifying citation. Thedisclosures of these publications, patents and published patentspecifications are hereby incorporated by reference into the presentdisclosure to describe more fully the state of the art to which thisinvention pertains.

General Techniques

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of organic chemistry, pharmacology,molecular biology (including recombinant techniques), cell biology,biochemistry, and immunology, which are within the skill of the art.Such techniques are explained fully in the literature, such as,“Molecular Cloning: A Laboratory Manual” Second Edition (Sambrook etal., 1989); “Oligonucleotide Synthesis” (M. J. Gait, ed., 1984); “AnimalCell Culture” (R. I. Freshney, ed., 1987); the series “Methods inEnzymology” (Academic Press, Inc.); “Handbook of ExperimentalImmunology” (D. M. Weir & C. C. Blackwell, eds.); “Gene Transfer Vectorsfor Mammalian Cells” (J. M. Miller & M. P. Calos, eds., 1987); “CurrentProtocols in Molecular Biology” (F. M. Ausubel et al., eds., 1987, andperiodicals) “Polymerase Chain Reaction” (Mullis et al., eds., 1994);and “Current Protocols in Immunology” (J. E. Coligan et al., eds.,1991).

DEFINITIONS

As used herein, certain terms may have the following defined meanings.As used in the specification and claims, the singular forms “a”, “an”and “the” include plural references unless the context clearly dictatesotherwise. For example, the term “a cell” includes a plurality of cells,including mixtures thereof. Similarly, use of “a compound” for treatmentor preparation of medicaments as described herein contemplates using oneor more compounds of this invention for such treatment or preparationunless the context clearly dictates otherwise.

As used herein, the term “comprising” is intended to mean that thecompositions and methods include the recited elements, but not excludingothers. “Consisting essentially of” when used to define compositions andmethods, shall mean excluding other elements of any essentialsignificance to the combination. Thus, a composition consistingessentially of the elements as defined herein would not exclude tracecontaminants from the isolation and purification method andpharmaceutically acceptable carriers, such as phosphate buffered saline,preservatives, and the like. “Consisting of” shall mean excluding morethan trace elements of other ingredients and substantial method stepsfor administering the compositions of this invention. Embodimentsdefined by each of these transition terms are within the scope of thisinvention.

Invention

We demonstrate that mucosal delivery of an immune dominant antigensecreted by a micro-organism such as preferably L. lactis, inducessuppression of local and systemic T-cell responses. Treatment resultedin an antigen-specific decrease of the proliferative capacity of thesplenocytes and inguinal lymph node cells, which was criticallydependent on the production of IL-10 and TGF-β and associated with asignificant induction of Foxp3+ regulatory T-cells. This approach ofantigen-delivering bacteria has the capacity for potentiating oraltolerance even in the setting of established hypersensitivity. Thus itis applicable for the treatment of celiac disease and other autoimmuneand/or allergic diseases. The efficacy of the invention was demonstratedin autoimmune and allergic disease mouse models, as well as in thecontext of immune inactivation of therapeutics.

A first aspect of the invention is a method for inducing immunetolerance to an antigen, comprising mucosal delivery of said antigen bya micro-organism.

Preferably the invention relates to the use of a micro-organism,preferably a non-pathogenic micro-organism, more preferably lactic acidbacterium or yeast, even more preferably a Lactococcus lactis secretingan antigen for the preparation of a medicament, medical food ornutraceutical for mucosal delivery to treat an immune response relateddisease in a patient, wherein said antigen is preferably continuouslypresent in said patient.

Preferably, said antigen is delivered by an antigen expressingmicro-organism. Preferably said antigen is delivered by an antigensecreting or antigen displaying micro-organism or an intracellularantigen. Thus, the invention encompasses embodiments wherein saidantigen is displayed at the surface of said antigen expressingmicro-organism or wherein said antigen is secreted, or said antigen isfreed upon digestion.

Preferably, the present invention relates to the use of an antigenexpressing micro-organism for the preparation of a medicament formucosal delivery to induce immune tolerance.

Preferably, said immune tolerance is induced in a patient. Said patientis preferably an animal. Said animal is preferably a mammal, andpreferably chosen from the group consisting of mouse, rat, pig, cow,sheep, horses and human. Preferably, said mammal is human. Preferably,said immune tolerance is mucosal tolerance.

Mucosa

Mucosa as used here can be any mucosa such as oral mucosa, rectalmucosa, urethral mucosa, vaginal mucosa, ocular mucosa, buccal mucosa,pulmonary mucosa and nasal mucosa. Mucosal delivery as used throughoutthe application encompasses the delivery to the mucosa. Oral mucosaldelivery includes buccal, sublingual and gingival routes of delivery.Accordingly, the present invention relates to method in which saidmucosal delivery is chosen from the group consisting of rectal delivery,buccal delivery, pulmonary delivery, ocular delivery, nasal delivery,vaginal delivery and oral delivery. Preferably, said mucosal delivery isoral delivery and said tolerance is oral tolerance.

Mucosal tolerance as used here throughout the application is theinhibition of specific immune responsiveness to an antigen in an animal(including humans), after that said animal has been exposed to saidantigen via the mucosal route. Preferably, said mucosal tolerance issystemic tolerance. The subsequent exposure of the antigen can be everyexposure known to the person skilled in the art, such as exposure byparenteral injection, by mucosal delivery, or by endogenous productionsuch as in the case of auto-antigens. Oral tolerance is the inhibitionof specific immune responsiveness to an antigen in an animal (includinghumans), after that said animal has been exposed to said antigen via theoral route. Low dose oral tolerance is oral tolerance induced by lowdoses of antigens, and is characterized by active immune suppression,mediated by cyclophosphamide sensitive regulatory T-cells that cantransfer tolerance to naive hosts. High dose oral tolerance is oraltolerance induced by high doses of antigens, is insensitive tocyclophosphamide treatment, and proceeds to induction of T cellhyporesponsiveness via anergy and/or deletion of antigen specificT-cells. The difference in sensitivity to cyclophosphamide can be usedto make a distinction between low dose and high dose tolerance (Strobelet al., 1983). Preferably, said oral tolerance is low dose oraltolerance as described by Mayer and Shao (2004b).

The present invention thus relates to a method or use as describedherein, wherein said induction of immune tolerance is at least 1.5,preferably 2, more preferably 3 times or more relative to before saidinduction. Alternatively, said antigen is tolerated at least 1.5, 2, 3times or more relative to before said induction. The induction of immunetolerance can be measured by methods known in the art. Preferably, saidinduction of immune tolerance can be measured by modulation of acytokine level in said animal. As such, the modulation can be anincrease of a cytokine level, for instance said increase of a cytokinelevel is at least 1.5, 2, 3 times or more relative to before saidinduction, e.g. IL-10 or TGF-β. Alternatively, said modulation is adecrease of the level of a particular cytokine level, for instance saiddecrease of the cytokine level is at least 1.5, 2, 3 times or morerelative to before said induction, e.g. IL-12, IL-17 and IFN-γ. Thecytokines which are modulated may be chosen from any relevant cytokines,preferably said cytokines are chosen from the group consisting of IL-2,IL-4, IL-5, IL-6, IL-10, IL-12, IL-13, IL-17, IL-23, TNF-α, IFN-γ,IFN-α, MCP-1, TGF-β, RANK-L and Flt3L.

Constructs, Delivery & Integration

In the present invention, the micro-organism delivers the antigen at theintended site, i.e. mucosa. The micro-organism expresses said antigen,after which the antigen is exposed on the cell surface or secreted.Hence, in a preferred embodiment the micro-organism, such as L. lactis,comprises an expression vector capable of expressing the heterologousantigen, e.g. the antigen used for inducing immune tolerance,intracellularly, secreted and/or such that the heterologous antigen isexposed on the cell surface under conditions present at the intendedmucosa, e.g. such as in the gastrointestinal tract. The micro-organism,e.g. L. lactis, can comprise expression vectors capable of expressingthe heterologous antigen intracellularly, secreted and/or such that theheterologous antigen is exposed on the cell surface to a degreesufficient to induce immune tolerance. As high a degree of expression aspossible without damaging the viability of the cell or the host to betreated is envisaged. With higher expression, less frequent and lowerdoses may be required for tolerance purposes. Naturally the dosageregime will not only depend on amount of antigen but also on antigentype and the presence or absence of other immunogenicity stimulating orsuppressing factors in the composition.

Usually, the expression system will comprise a genetic constructcomprising at least one nucleotide sequence encoding the desiredantigen, preferably operably linked to a promoter capable of directingexpression of the sequence in the hosting micro-organism. Suitably theantigen to be expressed can be encoded by a nucleic acid sequence thatis adapted to the preferred codon usage of the host. The construct mayfurther contain (all) other suitable element(s), including enhancers,transcription initiation sequences, signal sequences, reporter genes,transcription termination sequences, etc., operable in the selectedhost, as is known to the person skilled in the art. The construct ispreferably in a form suitable for transformation of the host and/or in aform that can be stably maintained in the host, such as a vector,plasmid or mini-chromosome. Suitable vectors comprising nucleic acid forintroduction into micro-organisms, e.g. bacteria, can be chosen orconstructed, containing appropriate regulatory sequences, includingpromoter sequences, terminator fragments, enhancer sequences, markergenes and other sequences as appropriate. Vectors may be plasmids, virale.g. ‘phage, or phagemid, as appropriate. For further details see, forexample, Molecular Cloning: a Laboratory Manual: 2nd edition, Sambrooket al., 1989, Cold Spring Harbor Laboratory Press. Many known techniquesand protocols for manipulation of nucleic acid, for example inpreparation of nucleic acid constructs, mutagenesis, sequencing,introduction of DNA into cells and gene expression, and analysis ofproteins, are described in detail in Short Protocols in MolecularBiology, Second Edition, Ausubel et al. eds., John Wiley & Sons, 1992.The disclosures of Sambrook et al. and Ausubel et al. are incorporatedherein by reference. In a preferred embodiment, the coding sequences forthe biologically active polypeptide and the antigen are contained in anoperon, i.e. a nucleic acid construct for multi-cistronic expression. Inan operon, transcription from the promoter results in a mRNA whichcomprises more than one coding sequence, each with its own suitablypositioned ribosome binding site upstream. Thus, more than onepolypeptide can be translated from a single mRNA. Use of an operonenables expression of the biologically active polypeptide and theantigen to be co-ordinated. More preferably, a food grade construct isused.

In an embodiment the present invention relates to stably transfectedmicro-organisms, i.e. micro-organisms in which the gene coding for theantigen has been integrated into the host's genome. Techniques forestablishing stably transfected micro-organisms are known in the art.For instance, the gene of interest may be cloned into the host's genomevia homologous recombination. Preferably, an essential gene of the hostis disrupted by the homologous recombination event, such as deletion ofthe gene, one or more amino acid substitutions leading to an inactiveform of the protein encoded by the essential gene, or to a frameshiftmutation resulting in a truncated form of the protein encoded by theessential gene. In an embodiment, the essential gene is the thyA gene. Apreferred technique is described in WO02/090551, which is specificallyincorporated herein in its entirety. The transforming plasmid can be anyplasmid, as long as it cannot complement the disrupted essential gene,e.g. thyA gene. The plasmid may be a self-replicating, preferablycarrying one or more genes of interest and one or more resistancemarkers, or the plasmid is an integrative plasmid. In the latter case,the integrative plasmid itself may be used to disrupt the essentialgene, by causing integration at the locus of the essential gene, e.g.thyA site, because of which the function of the essential gene, e.g. thethyA gene, is disrupted. Preferably, the essential gene, such as thethyA gene, is replaced by double homologous recombination by a cassettecomprising the gene or genes of interest, flanked by targeting sequencesthat target the insertion to the essential gene, such as the thyA targetsite. It will be appreciated that that these targeting sequences aresufficiently long and sufficiently homologous to enable integration ofthe gene of interest into the target site.

The genetic construct encoding the antigen of the invention may thus bepresent in the host cell extra-chromosomally, preferably autonomouslyreplicating using an own origin of replication, or may be integratedinto the microbial genomic DNA, e.g., bacterial or yeast chromosome,e.g., Lactococcus chromosome. In the latter case, a single or multiplecopies of the said nucleic acid may be integrated; the integration mayoccur at a random site of the chromosome or, as described above, at apredetermined site thereof, preferably at a predetermined site, such as,in a preferred non-limiting example, in the thyA locus of Lactococcus,e.g., Lactococcus lactis.

Hence, in an embodiment, the genetic construct encoding the antigen ofthe invention may further comprises sequences configured to effectinsertion of the said genetic construct into the genome, e.g., achromosome, of a host cell.

In an example, insertion of the genetic construct into particular siteswithin a genome, e.g. chromosome, of a host cell may be facilitated byhomologous recombination. For instance, the genetic construct theinvention may comprise one or more regions of homology to the said siteof integration within the genome e.g., a chromosome, of the host cell.The sequence at the said genome, e.g. chromosome, site may be natural,i.e., as occurring in nature, or may be an exogenous sequence introducedby previous genetic engineering.

For instance, the said region(s) of homology may be at least 50 bp,preferably at least 100 bp, e.g., at least 200 bp, more preferably atleast 300 bp, e.g., at least 400 bp, even more preferably at least 500bp, e.g., at least 600 by or at least 700 bp, still more preferably atleast 800 bp, e.g., at least 900 bp, or at least 1000 by or more.

In a preferred example, two regions of homology may be included, oneflanking each side of the relevant expression units present in thegenetic construct of the invention. Such configuration mayadvantageously insert the relevant sequences, i.e., at least the onesencoding and effecting the expression of the antigen of interest, inhost cells. Ways of performing homologous recombination, especially inbacterial hosts, and selecting for recombinants, are generally known inthe art.

Transformation methods of micro-organisms are known to the personskilled in the art, such as for instance protoplast transformation andelectroporation.

A high degree of expression can be achieved by using homologousexpression and/or secretion signals on the expression vectors present inthe micro-organism, e.g. L. lactis. Suitably expression regulatingsignals as present in the constructs in the Examples are useful. Otherexpression signals will be apparent to the person skilled in the art.The expression vector can be optimised for expression depending on themicro-organism, e.g. L. lactis, it is incorporated in. For instance,specific expression vectors that gave sufficient levels of expression inLactococcus, Lactobacillus lactis, casei and plantarum are known.Moreover, systems are known which have been developed for the expressionof heterologous antigens in the non-pathogenic, non-colonising,non-invasive food-grade bacterium Lactococcus lactis (see UK patentGB2278358B, which is incorporated herein by reference). A particularlypreferred construct according to the invention comprises the multi-copyexpression vector described in PCT/NL95/00135 (WO-A-96/32487), in whichthe nucleotide sequence encoding the antigen has been incorporated. Sucha construct is particularly suitable for expression of a desired antigenin a lactic acid bacterium, in particular in a Lactobacillus, at a highlevel of expression, and also can be used advantageously to direct theexpressed product to the surface of the bacterial cell. The constructs(e.g. of PCT/NL95/00135) may be characterised in that the nucleic acidsequence encoding the antigen is preceded by a 5′ non-translated nucleicacid sequence comprising at least the minimal sequence required forribosome recognition and RNA stabilisation. This can be followed by atranslation initiation codon which may be (immediately) followed by afragment of at least 5 codons of the 5′ terminal part of the translatednucleic acid sequence of a gene of a lactic acid bacterium or astructural or functional equivalent of the fragment. The fragment mayalso be controlled by the promoter. The contents of PCT/NL95/00135,including the differing embodiments disclosed therein, and all otherdocuments mentioned in this specification, are incorporated herein byreference. One aspect of the present invention provides a method whichpermits the high level regulated expression of heterologous genes in thehost and the coupling of expression to secretion. In a further preferredembodiment, the T7 bacteriophage RNA polymerase and its cognate promoterare used to develop a powerful expression system according toWO93/17117, which is incorporated herein by reference. Preferably theexpression plasmid is derived from pT1 NX.

A promoter employed in accordance with the present invention ispreferably expressed constitutively in the bacterium. The inventorsobserved that constitutive expression of the antigen resulted inincreased immune tolerance in contrast to inducible expression.Furthermore, the use of a constitutive promoter avoids the need tosupply an inducer or other regulatory signal for expression to takeplace. Preferably, the promoter directs expression at a level at whichthe bacterial host cell remains viable, i.e. retains some metabolicactivity, even if growth is not maintained. Advantageously then, suchexpression may be at a low level. For example, where the expressionproduct accumulates intracellularly, the level of expression may lead toaccumulation of the expression product at less than about 10% ofcellular protein, preferably about or less than about 5%, for exampleabout 1-3%. The promoter may be homologous to the bacterium employed,i.e. one found in that bacterium in nature. For example, a Lactococcalpromoter may be used in a Lactococcus. A preferred promoter for use inLactococcus lactis (or other Lactococci) is “P1” derived from thechromosome of Lactococcus lactis (Waterfield, N R, Lepage, R W F,Wilson, P W, et al. (1995). The isolation of lactococcal promoters andtheir use in investigating bacterial luciferase synthesis in Lactococcuslactis. Gene 165(1), 9-15). Another preferred promoter is the usp45promoter.

The nucleic acid construct or constructs may comprise a secretory signalsequence. Thus, in a preferred embodiment the nucleic acid encoding anantigen may provide for secretion of said antigen (by appropriatelycoupling a nucleic acid sequence encoding a single sequence to thenucleic acid sequence encoding the antigen). Ability of a bacteriumharbouring the nucleic acid to secrete the antigen may be tested invitro in culture conditions which maintain viability of the organism.Preferred secretory signal sequences include any of those with activityin Gram positive organisms such as Bacillus, Clostridium andLactobacillus. Such sequences may include the α-amylase secretion leaderof Bacillus amyloliquetaciens or the secretion leader of theStaphylokinase enzyme secreted by some strains of Staphylococcus, whichis known to function in both Gram-positive and Gram-negative hosts (see“Gene Expression Using Bacillus”, Rapoport (1990) Current Opinion inBiotechnology 1:21-27), or leader sequences from numerous other Bacillusenzymes or S-layer proteins (see pp 341-344 of Harwood and Cutting,“Molecular Biological Methods for Bacillus”, John Wiley & Co. 1990).Preferably, said secretion signal is derived from usp45 (Van Asseldonket al. 1993 Mol. Gen. Genet. 240:428-434). Preferably, said antigen isconstitutively secreted.

In an alternative embodiment, the coding sequences for the biologicallyactive polypeptide and the antigen are part of the same nucleic acidvector, or separate vectors, and are individually under the regulatorycontrol of separate promoters. The promoters may be the same ordifferent. A nucleic acid construct or vector comprising a codingsequence for a biologically active polypeptide and a coding sequence foran antigen wherein each coding sequence is under the control of apromoter for expression in a non-invasive host, e.g. Lactococcus,whether as an operon or not, is provided by a further aspect of thepresent invention.

Antigens

The sequence encoding the antigen can be obtained from any naturalsource and/or can be prepared synthetically using well known DNAsynthesis techniques. The sequence encoding the antigen can then (forinstance) be incorporated in a suitable expression vector to provide agenetic construct of the invention, which is then used to transform ortransfect the intended host. The recombinant thus obtained can then becultured, upon which the harvested cells can be used to formulate thecomposition, optionally after further purification and/or processingsteps, such as freeze-drying to form a powder.

An antigen can be any antigen known to the person skilled in the art. Anantigen as used here throughout the application is preferably anysubstance that provokes an immune response when introduced in the bodyof an animal, wherein said immune response can be T-cell mediated and/ora B-cell mediated response. The antigen may comprise a T-cell epitopeand/or a B-cell epitope. The length of the antigen is not particularlylimiting, provided said antigen can be expressed in the micro-organismof the invention. The antigen can be a protein or a part thereof, suchas a polypeptide or a peptide. The antigens according to the inventioninclude linear and/or conformational epitopes. T-cell mediated responsescover Th1, Th2 and/or Th17 responses. The antigen can be any antigen,such as, but not limited to allergens (including food allergens),allo-antigens, self-antigens, auto-antigens, and therapeutic moleculesor antigens that induce an immune response. Preferably, said antigen isinvolved in the induction of immune response related diseases. Even morepreferably, said antigen is involved in the induction of allergicasthma, multiple sclerosis, type I diabetes, autoimmune uveitis,autoimmune thyroiditis, autoimmune myasthenia gravis, rheumatoidarthritis, food allergy, celiac disease or graft versus host disease.

The inventors observed that the secreted immunodominant antigens of theinvention suppress systemic inflammatory T-cell responses, and thatthese antigens are necessary and sufficient for the induction of asignificant tolerogenic effect.

Regulatory T cells (Treg) play a critical role in the induction andmaintenance of oral tolerance. Induction of Treg is a major goal forimmunotherapy for several autoimmune, allergic and inflammatorydiseases. Current strategies for therapeutic induction ofantigen-specific suppressor cells face significant hurdles, and usuallyrequire strenuous techniques to isolate, handle and transfer adequatenumbers of regulatory cells. The micro-organism, e.g. L. lactis-antigendelivery system of the present invention circumvents these problems andeffectively induces antigen-specific Treg. In the present invention itwas demonstrated that induction of Treg can be achieved by exposing themucosal immune system to low doses of antigen. The exposure to low dosesof antigen is preferably a continued exposure. Hence, the presentinvention relates to antigens inducing and/or expanding Treg cells,preferably CD4⁺CD25⁺, CD4⁺CD25⁻ and CD8⁺ Treg cells.

It was further demonstrated in the present invention that the Treg cellswhich were induced and/or expanded by the antigens according to theinvention function through a TGF-β and/or IL-10 dependent mechanism.Previously evidence has been provided that TGF-β plays a critical rolein oral tolerance as well as in the development of peripheral inducedTreg. Accordingly, the present invention provides immunodominantantigens which stimulate endogenous TGF-β and/or IL-10 expression.

Moreover it was shown that antigen-specific TGF-β producing Th3 cellsdrive the differentiation of antigen-specific Foxp3⁺ regulatory cells inthe periphery. Furthermore TGF-β dependent conversion of peripheralCD4⁺CD25⁻ T cells into CD25⁺, CD45RB^(−/low) suppressor cells has beenreported. It was shown that oral tolerance induced by CTB-conjugated Agis associated with increase in TGF-β by the generation of bothFoxp3⁺CD25⁺ and both Foxp3⁺ and Foxp3−CD25⁻ CD4⁺ regulatory T Cells.These data suggest a key role for Foxp3⁺ ‘adaptive’ Treg in theinduction and maintenance of oral tolerance. We also show a significantmucosal Foxp3 induction. Moreover, the ‘mucosal’ induced regulatoryT-cell tends to be antigen specific as L. lactis alone is unable toinduce this Foxp3 upregulation within the GALT. Accordingly, the presentinvention relates preferably to Foxp3+ Treg cells.

The present invention further demonstrated that the Treg cells whichwere induced and/or expanded by the antigens according to the inventiondecreased inflammation, in particular in the spleen and inguinal lymphnode cells. Moreover, the IFN-γ and IL-12 production was decreased.Accordingly, the present invention provides immunodominant antigenswhich decrease endogenous IFN-γ and/or IL-12 production, and/orstimulate endogenous TGF-β and/or IL-10 expression. Moreover, thepresent invention relates to antigens reducing proliferation of spleenand/or inguinal lymph node cells. It will be appreciated that thepresent invention relates also to antigens suppressing inflammatoryantigen specific T cell response.

It will be appreciated that certain HLA-DQ isoforms are more commonlyassociated with certain autoimmune diseases. For instance, the chronicsmall intestinal inflammation that defines celiac disease ischaracterized by a loss of tolerance to ingested gluten peptides and isstrongly associated with a HLA-DQ2 or HLA-DQ8 restricted T-cellresponse. The expression of HLA-DQ2 or HLA-DQ8 is necessary for theexpression of celiac, and confer up to 40% of the genetic risk inWestern populations. One of the most important aspects in thepathogenesis of celiac is the activation of a T-helper 1 immuneresponse, which arises when antigen-presenting cells that expressHLA-DQ2/DQ8 molecules present gluten peptides to CD4+ T-cells.

DQ8 stands out because of its strong association with not only celiacdisease but also juvenile diabetes. It is also linked to HLA-DR allelesthat are implicated in RA and may increase risk. HLA-DQ is not spreaduniformly and certain populations are at increased risk; however thatrisk is often dependent on environment (gluten consumption) andincreasing prevelance of some diseases may be the result of shifts ofindividual from low-risk environments to higher risk environments.

The HLA DQ8 according to the invention is the serotypic representationof an DQA1:DQB1 haplotype. DQ8 represents the haplotypesDQA1*0301:DQB1*0302, DQA1*0302:DQB1*0302, or DQA1*0303:DQB1*0302haplotypes. These haplotypes are associated with some of the most commonautoimmune disease known. DQA1*0301:DQB1*0302 is the most frequent ofthese 3 haplotypes and represents about 80% of the global DQ8. Thepresent invention thus relates to antigens recognized via DQA1*0301:DQB1*0302, DQA1*0302:DQB1*0302, and/or DQA1*0303:DQB1*0302 haplotypes,referred to as “DQ8 epitope”.

HLA-DQ2 is expressed in more than 90% of people with celiac disease. HLADR3-DQ2 is the serotypic representation of a HLA-DRB1: DQA1:DQB1haplotype. DR3-DQ2 principally represents the haplotype DRB1*0301:DQA1*0501: DQB1*0201. It is relatively abundant in western hemisphere.DQ2 is encoded by DQB1*02 alleles in combination with other alphaalleles. The two most common DQ2 β chains are very similar. The presentinvention thus relates to antigens recognized via DQB1*0201, DQB1*0202and/or DQB1*0203 haplotypes, referred to as “DQ2 epitope”.

The present invention relates preferably to antigens which are derivedfrom glycoproteins. Preferably said antigens are derived from gliadin,preferably α-gliadin and/or hordein. The gliadins, which can besubdivided into the α-, γ-, and ω-gliadins, and hordein are well knownin the art, and their sequences are easily retrievable via public domainlibraries, such as NCBI. Preferably, said α-gliadin is derived fromTriticum, such as T. aestivum or T. turgidum.

The present invention demonstrates that CD4+ T cells recognize nativegluten peptides in the context of DQ2 or DQ8.

In an embodiment the present invention relates to the DQ8 epitope:

QYPSGQGSFQPSQQNPQA, corresponding to residues 203-220 of the sequenceretrievable via UniProtKB/TrEMBL entry Q9M4L6 (SEQ ID NO: 4).

Said native DQ8 epitope is preferably encoded by the nucleotide sequence5′-caa tac cca tca ggt caa ggt tca ttc caa cca tca caa caa aac cca caaget-3′. (SEQ ID NO: 3)

In an embodiment the present invention relates to the DQ2 epitope:

LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF, corresponding to residues 57-89 ofthe sequence retrievable via UniProtKB/TrEMBL entry Q9M4L6 (SEQ ID NO:8)

Said DQ2 epitope is preferably encoded by the nucleotide sequence

5′-tta caa tta caa cca ttc cca caa cca caa tta cca tac cca tta cca taccca caa cca caa tta cca tac cca caa cca caa cca ttc (SEQ ID NO: 7)

Antigens are commonly deamidated in the intestines by e.g. endogenoustissue trans-glutaminase. Deamidated antigens are more immune reactiveand readily recognized than antigens which are not deamidated. Thepresence of endogenous tissue trans-glutaminase is indifferent in casethe antigens are deamidated by other means. In an embodiment, thepresent invention relates to deamidated antigens, encoded by nucleotidesequences in which codons for glutamine residues in epitopes arepreferably replaced by codons for glutamic acid residues.

In particular, the present invention relates to deamidated DQ8 epitope

QYPSGEGSFQPSQENPQA. (SEQ ID NO: 2)

Said deamidated DQ8 epitope is preferably encoded by the nucleotidesequence 5′-caa tac cca tca ggt gaa ggt tca ttc caa cca tca caa gaa aaccca caa get-3′. (SEQ ID NO: 1)

In particular, the present invention relates to deamidated DQ2 epitope

(SEQ ID NO: 6) LQL QPF PQP ELP YPQ PQL PYP QPE LPY PQP QPF

Said deamidated DQ2 epitope is preferably encoded by the nucleotidesequence 5′-tta caa tta caa cca ttc cca caa cca gaa tta cca tac cca ttacca tac cca caa cca gaa tta cca tac cca caa cca caa cca ttc (SEQ ID NO:5)

It was further demonstrated that the presence of additional sequences,such as a tag, to the epitope sequences did not influence the immuneresponse. Accordingly, in further embodiments, said epitope may comprisefurther amino acids, such as for instance 50 amino acids, 43, 30, 25,20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1amino acid(s). Hence, the present invention relates to DQ8 epitopescomprising at most 50 additional amino acids. In a further embodiment,the present invention relates to the amino acid sequenceGAPVPYPDPLEPRQYPSGEGSFQPSQENPQA (SEQ ID NO: 16), comprising a DQ8epitope and an e-tag (GAPVPYPDPLEPR (SEQ ID NO: 31)).

Immune Response

An immune response related disease as used here is a disease caused byan unwanted immune response of the body against an antigen, whereby saidantigen can be either a heterologous antigen or an auto-antigen. Immuneresponse related diseases include, but are not limited to allergicreaction including food allergy, celiac disease, allergic asthma,autoimmune uveitis, autoimmune thyroiditis, autoimmune myastheniagravis, rheumatoid arthritis, type I diabetes and multiple sclerosis.Immune response related diseases also include unwanted immune reactionssuch as graft versus host disease or immune activation of medicationsuch as the antibody production against non endogenous Factor VIII.Preferably, the disease is selected from the group consisting ofallergic asthma, food allergy, celiac disease, type I diabetes andimmune inactivation of therapeutics. It will thus be appreciated thatimmune response related diseases include, but are not limited toallergic reaction including food allergy, celiac disease, allergicasthma, autoimmune uveitis, autoimmune thyroiditis, autoimmunemyasthenia gravis, rheumatoid arthritis, type I diabetes and multiplesclerosis. Immune response related diseases also include unwanted immunereactions such as graft versus host disease or immune-activation ofmedication such as the antibody production against non endogenous FactorVIII. Preferably, the disease is selected from the group consisting ofallergic asthma, food allergy, celiac disease, graft versus hostdisease, type I diabetes and immune inactivation of therapeutics.

According to the present invention the term “immunodominant” relates tothe principle antigens inducing an immune response.

In view of the above, it will thus be appreciated that the presentinvention relates to method or use as described herein, wherein saidmethod or use is therapeutic and/or prophylactic.

A further aspect of the invention relates to a method for inducingimmune tolerance to an antigen, comprising mucosal delivery of saidantigen by a micro-organism in combination with mucosal delivery of animmune-modulating compound producing micro-organism. Theimmune-modulating compound and the antigen may be delivered by the samemicro-organism, or it may be a different micro-organism.

Medicament and Administration

Compound means any chemical of biological compound or complex, includingsimple or complex organic and inorganic molecules, peptides,peptido-mimetics, proteins, protein complexes, antibodies,carbohydrates, nucleic acids or derivatives thereof. Animmune-modulating compound is a compound that modifies the function ofthe immune system. An immune-modulating compound as used here is atolerance inducing compound; tolerance induction can be obtained, as anon-limiting example, in a direct way by inducing regulatory T-cellssuch as Treg, Tr1 or Th3, or by shifting the Th1/Th2 balance towards Th1or Th2, or by inhibiting Th17, or in an indirect way, by activation ofimmature dendritic cells to tolerizing dendritic cells and/or inhibitingTh2 immune response inducing expression of “co-stimulation” factors onmature dendritic cells. Immune-modulating and immune-suppressingcompounds are known to the person skilled in the art and include, butare not limited to bacterial metabolites such as spergualin, fungal andstreptomycal metabolites such as tacrolimus, rapamicin or ciclosporin,immune-suppressing cytokines such as IL-4, IL-10, IFNα TGFβ (asselective adjuvant for regulatory T-cells) Flt3L, TSLP, CTB and Rank-L(as selective tolerogenic DC inducers antibodies and/or antagonist suchas anti-CD40L, anti-CD25, anti-CD20, anti-IgE, anti-CD3, anti-IL-6 (orIL6R) and proteins, peptides or fusion proteins such as the CTL-4 Ig orCTLA-4 agonist fusion protein.

Thus, the immune-modulating compound can be any immune-modulatingcompound known to the person skilled in the art. Preferably, saidimmune-modulating compound is an immune-suppressing compound, even morepreferably said compound is an immune-suppressing cytokine or antibody.Preferably, said immune-suppressing cytokine is a tolerance-enhancingcytokine or antibody. Immune-suppressing cytokines are known to theperson skilled in the art, and include, but are not limited to IL-4,IL-10, IFN-α and TGF-β, as selective adjuvant for regulatory T-cells;and Flt3L, TSLP, CTB and Rank-L, as selective tolerogenic DC inducers.Preferably, said immune-suppressing cytokine is selected from the groupconsisting of IL-4, IL-10, IFNα and Flt3L. It will be appreciated by theperson skilled in the art that the present invention also relates tofunctional homologues thereof. A functional homologue connotes amolecule having essentially the same or similar, at least for theintended purposes, function, but can differ structurally. Mostpreferably, said immune-suppressing tolerance-enhancing cytokine isIL-10, or a functional homologue thereof. Preferably, saidimmune-suppressing antibody is chosen from the group consisting ofanti-IL-2, anti-IL12, anti-IL6, anti-IFN-γ.

Delivery as used here means any method of delivery known to the personskilled in the art and includes, but is not limited to, coated ornon-coated pharmaceutical formulations of the compound to deliver,capsules, liposomes, oil bodies, polymer particles comprising orcarrying the compound to deliver or micro-organisms secreting,displaying or accumulating the compound to deliver, optionally inpresence of compounds that may enhance mucosal delivery and/or mucosaluptake.

Compounds or compositions described herein may be administered in pureform, combined with other active ingredients, or combined withpharmaceutically acceptable nontoxic excipients or carriers. Oralcompositions will generally include an inert diluent carrier or anedible carrier. Pharmaceutically compatible binding agents, and/oradjuvant materials can be included as part of the composition. Tablets,pills, capsules, troches, enema and the like can contain any of thefollowing ingredients, or compounds of a similar nature: a binder suchas microcrystalline cellulose, gum tragacanth or gelatin; an excipientsuch as starch or lactose, a dispersing agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate; aglidant such as colloidal silicon dioxide; a sweetening agent such assucrose or saccharin; or a flavoring agent such as peppermint, methylsalicylate, or orange flavoring. When the dosage unit form is a capsule,it can contain, in addition to material of the above type, a liquidcarrier such as fatty oil. In addition, dosage unit forms can containvarious other materials that modify the physical form of the dosageunit, for example, coatings of sugar, shellac, or enteric agents.Further, syrup may contain, in addition to the active compounds, sucroseas a sweetening agent and certain preservatives, dyes, colorings, andflavorings. It will be appreciated that the form and character of thepharmaceutically acceptable carrier is dictated by the amount of activeingredient with which it is to be combined, the route of administrationand other well-known variables. The carrier(s) must be “acceptable” inthe sense of being compatible with the other ingredients of theformulation and not deleterious to the recipient thereof.

Alternative preparations for administration include sterile aqueous ornonaqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are dimethylsulfoxide, alcohols, propylene glycol,polyethylene glycol, vegetable oils such as olive oil and injectableorganic esters such as ethyl oleate. Aqueous carriers include mixturesof alcohols and water, buffered media, and saline. Intravenous vehiclesinclude fluid and nutrient replenishers, electrolyte replenishers, suchas those based on Ringer's dextrose, and the like. Preservatives andother additives may also be present such as, for example,antimicrobials, anti-oxidants, chelating agents, inert gases, and thelike. Various liquid formulations are possible for these deliverymethods, including saline, alcohol, DMSO, and water based solutions.

Preferably said antigen and/or said immune-suppressing cytokine isexpressed in low amounts, preferably 0.1 μg or lower per dose bacteriaadministered in a mice experimental setting, such amounts to betranslated in a human disease setting.

The terms “treatment”, “treating”, and the like, as used herein includeamelioration or elimination of a developed mental disease or conditiononce it has been established or alleviation of the characteristicsymptoms of such disease or condition. As used herein these terms alsoencompass, depending on the condition of the patient, preventing theonset of a disease or condition or of symptoms associated with a diseaseor condition, including reducing the severity of a disease or conditionor symptoms associated therewith prior to affliction with said diseaseor condition. Such prevention or reduction prior to affliction refers toadministration of the compound or composition of the invention to apatient that is not at the time of administration afflicted with thedisease or condition. “Preventing” also encompasses preventing therecurrence or relapse-prevention of a disease or condition or ofsymptoms associated therewith, for instance after a period ofimprovement. It should be clear that mental conditions may beresponsible for physical complaints. In this respect, the term“treating” also includes prevention of a physical disease or conditionor amelioration or elimination of the developed physical disease orcondition once it has been established or alleviation of thecharacteristic symptoms of such conditions.

As used herein, the term “medicament” also encompasses the terms “drug”,“therapeutic”, “potion” or other terms which are used in the field ofmedicine to indicate a preparation with therapeutic or prophylacticeffect.

It will be appreciated that the compound of the invention, i.e. theantigen, is delivered or expressed in a therapeutically effectiveamount. As used herein, the term “therapeutically effective amount” ismeant to refer to an amount of a compound or composition of the presentinvention that will elicit a desired therapeutic or prophylactic effector response when administered according to the desired treatmentregimen. It is observed that when the immune-dominant antigen iscontinuously present, the inflammatory antigen specific cell response iseven reduced further. This reduction is significantly larger compared toadministration of the antigen as such, the micro-organism as such, orthe non-continuous presence of the antigen. The term “continuouslypresent” or “continued presence” according to the invention relates tothe constant or uninterrupted presence of an antigen according toinvention at the intended mucosal site, e.g. the site of inflammation.The presence of the antigen can be measured by techniques well known inthe art, such as PCR, ELISA or immune precipitation techniques, such asfor instance detailed in the examples section and supra. Moreover, thepresence of L. lactis may be a measure of the presence of the antigen.Also, the effects caused by the antigen may be a measure of the presenceof the antigen, such as, for instance, the presence or increase ofendogenous TGF-β or IL-10 levels, or a decrease of IFN-γ or IL-12levels, or the presence of Treg cells, such as described herein, or adecrease of the proliferative capacity of the splenocytes and draininglymph node cells. It will thus be appreciated that the levels of theantigen may vary, while the antigen is still considered to becontinuously present.

Preferably the compound or composition is provided in a unit dosageform, for example a tablet, capsule, enema or metered aerosol dose, sothat a single dose is administered to the subject, e.g. a patient.

The active ingredients may be administered from 1 to 6 times a day,sufficient to exhibit the desired activity. These daily doses can begiven as a single dose once daily, or can be given as two or moresmaller doses at the same or different times of the day which in totalgive the specified daily dose. Preferably, the active ingredient isadministered once or twice a day. For instance, one dose could be takenin the morning and one later in the day.

In all aspects of the invention, the daily maintenance dose can be givenfor a period clinically desirable in the patient, for example from 1 dayup to several years (e.g. for the mammal's entire remaining life); forexample from about (2 or 3 or 5 days, 1 or 2 weeks, or 1 month) upwardsand/or for example up to about (5 years, 1 year, 6 months, 1 month, 1week, or 3 or 5 days). Administration of the daily maintenance dose forabout 3 to about 5 days or for about 1 week to about 1 year is typical.Other constituents of the liquid formulations may include preservatives,inorganic salts, acids, bases, buffers, nutrients, vitamins, or otherpharmaceuticals.

The micro-organism delivering the antigen may be delivered in a dose ofat least 10⁴ colony forming units (cfu) to 10¹² cfu per day, preferablybetween 10⁶ cfu to 10¹² cfu per day, most preferably between 10⁹ cfu and10¹² cfu per day. In accordance with the method as described in Steidleret al. (Science 2000), the antigen and possibly the immuno-modulatingcompound of e.g. of 10⁹ cfu is secreted to at least 1 ng to 100 ng.Through ELISA, known to a person skilled in the art, the skilled personin the art can calculate the range of secretion of antigen in relationto any other dose of cfu.

The antigen may be delivered in a dose inducing a low-dose response.Preferably, said antigen is delivered in a dose of at least 10 fg to 500μg per day, preferably between 1 pg and 250 μg per day, more preferablybetween 100 pg and 200 μg per day, or preferably 1 ng and 150 μg, ormore preferably 10 ng and 125 μg per day, even more preferably 100 ngand 100 μg per day, even more preferably 1 μg and 90 μg per day and mostpreferably between 10 μg and 75 μg per day, such as, for instance, 25μg, 30 μg, 40 μg, 50 μg, 60 μg or 70 μg per day.

Preferably the compounds or composition is provided in a unit dosageform, for example a tablet, solution, capsule or metered aerosol dose,so that a single dose is administered to the subject, e.g. a patient.

Depending on the mode of administration, e.g. oral, or any of the onesdescribed above, the man skilled in the art knows how to define orcalculate the actual dose to be administered to a patient. The personskilled in the art will be knowledgeable to adjust the doses dependingon the patient, micro-organism, vector et cetera.

Compounds of the present invention also may take the form of apharmacologically acceptable salt, hydrate, solvate, or metabolite.Pharmacologically acceptable salts include basic salts of inorganic andorganic acids, including but not limited to hydrochloric acid,hydrobromic acid, sulphuric acid, phosphoric acid, nitric acid,methanesulphonic acid, ethanesulfonic acid, p-toluenesulfonic acid,naphtalenesulfonic acid, malic acid, acetic acid, oxalic acid, tartaricacid, citric acid, lactic acid, fumaric acid, succinic acid, maleicacid, salicylic acid, benzoic acid, phenylacetic acid, mandelic acid andthe like. When compounds of the invention include an acidic function,such as a carboxy group, then suitable pharmaceutically acceptablecation pairs for the carboxy group are well known to those skilled inthe art and include alkaline, alkaline earth, ammonium, quaternaryammonium cations and the like.

Micro-Organism

The micro-organism according to the invention can be any micro-organism,including bacteria, yeasts or fungi, suitable for mucosal delivery.Preferably, said micro-organism is a non pathogenic micro-organism, evenmore preferably said micro-organism is a probiotic micro-organism.Probiotic organisms are known to the person skilled in the art.Probiotic organisms include, but are not limited to, bacteria such asLactobacillus sp., Lactococcus sp. and yeasts such as Saccharomycescerevisiae subspecies boulardii. Preferably, said bacterium is a lacticacid bacterium; Even more preferably, said lactic acid bacterium ischosen from the group consisting of Lactobacillus, Leuconostoc,Pediococcus, Lactococcus, Streptococcus, Aerococcus, Carnobacterium,Enterococcus, Oenococcus, Teragenococcus, Vagococcus, and Weisella. Inone further preferred embodiment, said micro-organism is Lactococcuslactis. In another preferred embodiment said lactic acid bacterium is aLactobacillus sp. In another preferred embodiment, said micro-organismis Saccharomyces cerevisiae, even more preferably said yeast isSaccharomyces cerevisiae subsp. Boulardii.

Most preferably said probiotic micro-organism is a lactic acidbacterium, as delivery of heterologous proteins (i.e. non Lactic acidbacterial proteins) by lactic acid bacteria into the mucosa, includingboth oral and vaginal delivery, has been described (Steidler andRottiers, 2006; Liu et al., 2006), which makes these lactic acidbacteria extremely suitable for delivery of said antigen and possiblysaid immune-suppressing compound. L. lactis is a non-pathogenic,non-invasive, non-colonizing gram-positive bacterium. A variety ofgenetically modified L. lactis strains is generated for local synthesisand delivery of immunomodulatory proteins to the intestinal mucosa.Furthermore, a biological containment system is established which makesclinical application of genetically engineered L. lactis a feasiblestrategy.

In one preferred embodiment said micro-organism is a Lactococcus lactisthyA mutant. A specially preferred embodiment uses a Lactococcus lactisthyA mutant, wherein the gene encoding the antigen has been used todisrupt the thyA gene.

Nutraceuticals & Medical Foods

It will be appreciated that the compounds and compositions of theinvention may be used as nutraceuticals, functional or medical food, oras additives in said nutraceuticals, functional or medical food. Anotherembodiment provides a food or beverage, preferably fit for humanconsumption, which is comprised of a nutraceutical and a flavoringagent, wherein the nutraceutical is comprised of an extract from anagricultural product.

Nutraceuticals, whether in the form of a liquid extract or drycomposition, are edible and may be eaten directly by humans, but arepreferably provided to humans in the form of additives or nutritionalsupplements e.g., in the form of tablets of the kind sold in health foodstores, or as ingredients in edible solids, more preferably processedfood products such as cereals, breads, tofu, cookies, ice cream, cakes,potato chips, pretzels, cheese, etc., and in drinkable liquids e.g.,beverages such as milk, soda, sports drinks, and fruit juices. Thus, inone embodiment a method is provided for enhancing the nutritional valueof a food or beverage by intermixing the food or beverage with anutraceutical in an amount that is effective to enhance the nutritionalvalue of the food or beverage.

Another embodiment provides a method for enhancing the nutritional valueof a food or beverage which comprises intermixing a food or a beveragewith a nutraceutical to produce a nutritionally-enhanced food orbeverage, wherein the nutraceutical is intermixed in an amount effectiveto enhance the nutritional value of the food or beverage, wherein thenutraceutical is comprised of an extract from a crop comprising theantigens of the present invention, and wherein thenutritionally-enhanced food or beverage may further comprise a flavoringagent. Preferred flavoring agents include sweeteners such as sugar, cornsyrup, fructose, dextrose, maltodextrose, cyclamates, saccharin,phenyl-alanine, xylitol, sorbitol, maltitol, and herbal sweeteners e.g.,Stevia.

The nutraceuticals described herein are intended for human consumptionand thus the processes for obtaining them are preferably conducted inaccordance with Good Manufacturing Practices (GMP) and any applicablegovernment regulations governing such processes. Especially preferredprocesses utilize only naturally derived solvents. The nutraceuticalsdescribed herein preferably contain relatively high levels ofhealth-enhancing substances Nutraceuticals may be intermixed with oneanother to increase their health-enhancing effects.

In contrast to nutraceuticals, the so-called “medical foods” are notmeant to be used by the general public and are not available in storesor supermarkets. Medical foods are not those foods included within ahealthy diet to decrease the risk of disease, such as reduced-fat foodsor low-sodium foods, nor are they weight loss products. A physicianprescribes a medical food when a patient has special nutrient needs inorder to manage a disease or health condition, and the patient is underthe physician's ongoing care. The label must clearly state that theproduct is intended to be used to manage a specific medical disorder orcondition. An example of a medical food is nutritionally diverse medicalfood designed to provide targeted nutritional support for patients withchronic inflammatory conditions. Active compounds of this product arefor instance one or more of the compounds described herein. Functionalfoods may encompass those foods included within a healthy diet todecrease the risk of disease, such as reduced-fat foods or low-sodiumfoods, or weight loss products. Hence, the present inventioncontemplates a food or beverage comprising a nutraceutical according tothe invention.

Those skilled in the art will appreciate that numerous changes andmodifications can be made to the preferred embodiments of the inventionand that such changes and modifications can be made without departingfrom the spirit of the invention. It is, therefore, intended that theappended claims cover all such equivalent variations as fall within thetrue spirit and scope of the invention.

In addition, all terms used in the description of compounds of thepresent invention have their meaning as is well known in the art.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Oral feeding of LL-OVA significantly reduces DTH responses.Balb/c mice were sensitized by s.c. injection of OVA/CFA on days 0 andreceived a boost immunization of OVA/IFA on day 21. Mice were orallytreated with BM9, LLpTREX1, LL-OVA and 1 μg OVA on days 7-11, 14-18,21-25 and 28-31. On day 31, mice were challenged with 10 μg Ova In 10 μlsaline in the auricle of the ears. The DTH responses were expressed asthe difference in ear thickness before and after the OVA challenge forboth ears 24 h postchallenge.

FIG. 2: Oral feeding of LL-OVA significantly reduces the OVA-specificproliferation (A) and IFN-γ (B), IL-6 (C) and IL-10 (D) production ofbulk splenocytes. Balb/c mice were sensitized by s.c. injection ofOVA/CFA on days 0 and received a boost immunization of OVA/IFA on day21. Mice were orally treated with BM9, LLpTREX1 and LL-OVA on days 21-25and 28-31. On day 31, bulk splenocytes were isolated and tested forOVA-specific proliferation, which is expressed as the mean cpm±SEM atdifferent OVA concentrations, and for IFN-γ, IL-6 and IL-10 productionafter 72-hour ex vivo stimulation with 100 μg/ml OVA.

FIG. 3: Oral feeding of LL-OVA significantly reduces the OVA-specificproliferation of CD4+ splenic T cells. Balb/c mice were sensitized bys.c. injection of OVA/CFA on days 0 and received a boost immunization ofOVA/IFA on day 21. Mice were orally treated with BM9 (A), LLpTREX1 (B)and LL-OVA (C) on days 21-25 and 28-31. On day 31, bulk splenocytes wereisolated and OVA-specific proliferation of CD4+ splenic T cells by CFSEand CD4-APC labelling and flow cytometric analysis after 90 h ex vivorestimulation with 100 μg/ml OVA.

FIG. 4: CD4+ T cells of LL-OVA treated mice transfer tolerance to naïverecipients. Balb/c mice were sensitized by s.c. injection of OVA/CFA ondays 0 and received a boost immunization of OVA/IFA on day 21. Mice wereorally treated with BM9, LLpTREX1 and LL-OVA on days 21-25 and 28-31. Onday 31, CD4+ splenic T cells were isolated and tested for tolerancetransfer capacity. Transfer of tolerance by CD4+ splenic T cells fromLL-OVA and LL-pTREX treated mice to the naïve recipients was assessed bysensitizing and challenging the latter for a DTH response, that isexpressed as the difference in ear thickness before and after the OVAchallenge for both ears 24 h postchallenge.

FIG. 5: NOD AB^(o) DQ8 transgenic mice were immunized by s.c. injectionof 100 μg eDQ8d in CFA at day 1. Mice were orally treated with LL-eDQ8dor LL-pT1NX at days 1-10. Control mice received BM9. At day 10, micewere challenged with 10 μg eDQ8d in 10 μl saline in the auricle of theear. DTH responses are expressed as the mean in increase 24 hours afterinjection, following subtraction of ear-thickness before eDQ8dchallenge. Results summarize data of 3 independent experiments including6 mice per group.

FIG. 6: After the DTH measurements, spleens (A) and inguinal lymph nodes(B) of the BM9 (control), LL-pT1NX and LL-eDQ8d groups were isolated andex vivo restimulated with 50 μg eDQ8d peptide. eDQ8d-specificproliferative response of bulk splenocytes (p=0.048) and inguinal lymphnode cells (p=0.0022) were expressed as the mean cpm.

FIG. 7: Cytokine measurements in the supernatant of spleen (A) andinguinal lymph node cells (B) were performed 24 hours afterrestimulation. Results are means of cytokine secretion in pg/mlrepresentative of at least two individual experiments.

FIG. 8: Decreased splenic eDQ8d-specific proliferation depends on IL-10and TGF-β.

EXAMPLES Example A Induction of OVA-Specific Tolerance by GeneticallyModified Lactococcus lactis Delivering OVA to OVA-Sensitized Wild TypeMice Introduction

For this purpose we genetically engineered OVA secreting LL (LL-OVA) andevaluated the induction of systemic tolerance in a therapeutic model forautoimmunity/allergy, namely the OVA immunization model.

Materials and Methods

Bacteria and media: The Lactococcus lactis MG1363 (LL) strain wasgenetically modified and used throughout this study. Bacteria werecultured in GM17E medium consisting of M17broth (Difco Laboratories,Detroit, Mich.) supplemented with 0.5% glucose and 5 μg/ml erythromycin(Abbott). Stock suspensions of LL strains were stored at −20° C. in 50%glycerol in GM17E medium. Stock suspensions were diluted 500-fold inGM17E medium and incubated at 30° C. overnight. Within 16 h they reacheda saturation density of 2×10⁹ colony forming units (CFU) per ml.Bacteria were harvested by centrifugation and resuspended in BM9 mediumat 2×10¹⁰ bacteria/ml. Each mouse received 100 μl of this suspensiondaily through an intragastric catheter.

Plasmids: The mRNA sequence encoding Gallus gallus Ovalbumin wasretrieved from Genbank (accession number AY223553) and from publisheddata. Total RNA was isolated from chicken uterus and cDNA wassynthesized using 2 μg total RNA, 2 μM oligo dT primers (PromegaCorporation Benelux, Leiden, The Netherlands), 0.01 mM DTT(Sigma-Aldrich, Zwijndrecht, The Netherlands), 0.5 mM dNTP (Invitrogen,Merelbeke, Belgium), 20 U Rnasin (Promega Incorporation Benelux) and 100U superscript II reverse transcriptase (Invitrogen) in a volume of 25μl. An OVA cDNA fragment was amplified by Polymerase Chain Reaction(PCR) using the following primers: forward5′-GGCTCCATCGGTGCAGCAAGCATGGAATT-3′ (SEQ ID NO: 9) and reverse5′-ACTAGTTAAGGGGAAACACATCTGCCAAAGAAGAGAA-3′(SEQ ID NO: 10). Reactionconditions were 94° C. for 2 min followed by 30 cycles at 94° C. for 45seconds, 62° C. for 30 seconds and 72° C. for 90 seconds. The amplifiedfragment was fused to the Usp45 secretion signal of the erythromycinresistant pT1 NX vector, downstream of the lactococcal P1 promotor17.MG1363 strains transformed with plasmids carrying OVA cDNA weredesignated L. lactis secreting OVA (LL-OVA). The L. lactis-pTREX1, whichis MG1363 containing the empty vector pTREX1, served as control(LL-pTREX).

Mice: Seven-week old female Balb/c mice were obtained from Charles RiverLaboratories (Calco, Italy) and were housed in a conventional animalfacility under specific pathogen-free conditions. The animal studieswere approved by the Ethics Committee of the Department for MolecularBiomedical Research at Ghent University (file No. 07/029).

Antigen: Intact, LPS-free OVA grade V protein was used as antigen in allexperiments (Sigma Aldrich).

Immunization of mice and induction of oral tolerance: Balb/c mice wereimmunized by s.c. injection of 100 μg OVA in 100 μl of a 1:1 mixture ofCFA (Difco, BD Bioscience, Erembodegem, Belgium) and saline solution atthe base of the tail on the first day. LL-OVA, LL-pTREX1 or 1 μgpurified OVA dissolved in 100 μl BM9 were administered daily on days7-11, 14-18, 21-25 and 28-31 (regime 1), and on days 21-25 and 28-31(regime 2). Control mice received only BM9. Antigen or bacterialsuspensions were introduced into the stomach using an 18-gauge stainlessanimal feeding needle. On day 21, a boost immunization was given by s.c.injection of 100 μg OVA in 100 μl of a 1:1 mixture of IFA(Sigma-Aldrich). Tolerance induction was assessed by DTH responses,measurement of cytokines and OVA-specific proliferation, and adoptivetransfer experiments.

Delayed-type Hypersensitivity responses: Antigen-specific DTH responseswere assessed by injection of OVA on day 31. Twenty-four hours later DTHmeasurements were performed. For measurement of antigen-specific DTHresponses, mice were challenged with 10 μg OVA in 10 μl saline in theauricle of the ear. Ear swelling, defined as the increase in earthickness due to challenge, was measured in a blinded fashion 24 h afterchallenge using a digital micrometer (Conrad, Belgium). The DTHresponses were expressed as the difference in ear thickness before andafter the OVA challenge for both ears.

OVA-specific proliferation and cytokine assays: On day 39, the spleenswere harvested and the splenocytes were assessed for OVA-specificproliferation and cytokine production. Single cell suspensions ofspleens were prepared by passing the cells through 70-μm cell strainers(Becton/Dickinson Labware). Erythrocytes in the cell suspensions werelysed by incubation with red cell lysis buffer. CD4+ T cells wereenriched using CD4+ T cell isolation kit and midiMACS columns (MiltenyiBiotec, Germany).

To assay proliferation of total splenocyte populations, 2×10⁵ cells werecultured in 96-well U-bottom plates in a total volume of 200 μl completemedium [i.e. RPMI-1640 containing 10% fetal calf serum (FCS), 10 U/mlpenicillin, 10 μg/ml streptomycin, 2 mM L-glutamax, 0.4 mM sodiumpyruvate] either alone or with OVA, added at concentrations ranging from1.2 to 100 μg/ml. The proliferation was further assessed by 5,6-CFSElabelling (Invitrogen, Merelbeke, Belgium). The splenocytes wereresuspended in PBS at 10⁷/ml and incubated in a final concentration of10 μM CFSE for 12 min at 37° C. Labelled cells were washed twice withice-cold complete medium before being cultured at 2×10⁵ cells in 96-wellU-bottom plates in a total volume of 200 μl complete medium with 100μg/ml OVA. After 90 h of culture at 37° C. and 5% CO₂ in a humidifiedincubator, the cells were harvested and the cells were stained withallophycocyanin-labeled anti-CD4 (BD, Biosciences) and proliferation wasdetermined using flow cytometry (FACSCanto, BD Biosciences).

To assay proliferation of CD4+ T cells, 2×10⁵ cells CD4+ T cells werecultured in 96-well U-bottom plates with mitomycin C treated-OVA loadedsplenocytes, acting as antigen presenting cells, at ratios 1/1, 1/0.3,1/0.1, 1/0.03 and 1/0 in a total volume of 200 μl complete medium. Cellswere cultured for 90 h at 37° C. and 5% CO₂ in a humidified incubator.For proliferation assays, 1 μCi/well [3H]-thymidine was added for thelast 18 h of culture, DNA was harvested on glass fibre filter mats(Perkin Elmer, Boston, USA), and DNA-bound radioactivity was measured ona scintillation counter (Perkin Elmer). For cytokine measurements,supernatants of the cell cultures used in the different proliferationassays were collected after 72 h of culture and frozen at −20° C.Cytokine production was quantified using the Mouse Flex Set CytometricBead Array (BD Biosciences, Mountain View, Calif., USA).

Adoptive transfer experiments: On day 39, the spleens were collectedfrom the treatment groups. Single cell suspensions were obtained bymincing the spleens and straining them through 70-μm cell strainers(Becton/Dickinson Labware). The cell suspensions were enriched for CD4+T cells, as described above. CD4+-enriched cells were adoptivelytransferred to naïve BALB/c acceptor mice by the i.v injection of 1×10⁶CD4+ T cells. One day after adoptive transfer, all mice were sensitizedby injection 100 μg OVA/25 μl saline/25 μl IFA (Sigma-Aldrich) s.c. atthe tail base, and 5 days thereafter, mice were challenged according tothe DTH protocol described above.

Statistical analysis: Significance of differences between groups inear-thicknesses and cytokine measurements were tested using one-wayANOVA. Statistical significance is indicated as * (p<0.05) or **(p<0.01).

Results LL-OVA Significantly Enhance the Tolerance-Inducing Capacity inOVA Immunization Model Compared to Free OVA.

To study the induction of oral tolerance, mice were orally fed asdescribed above. Administration of LL-OVA to OVA-sensitized BALB/c miceled to a significant decrease in DTH response compared to the sensitizedcontrol mice (BM9 group) and mice treated with LL-pTREX1 or 1 μgpurified OVA (FIG. 1)

These data were accompanied by a significant decreased proliferativecapacity and IFN-γ, IL-10 and IL-6 production (FIG. 2) of the bulksplenocytes of LL-OVA treated mice as compared to BM9 orLL-pTREX1-treated groups.

LL-OVA Enhances Oral Tolerance Via CD4+ T Cells.

To assess whether CD4 T cells mediate the induction of oral tolerance,the OVA-specific proliferative CD4 T cell response in the splenocyteswas studied. Flow cytometry demonstrated that only 0.8% of the CD4+splenic T cells proliferate after OVA restimulation in the LL-OVA groupcompared to 4.5% and 11.6% in the BM9 and LL-pTREX1 groups (FIG. 3).Furthermore, adoptive transfer CD4+ splenic T cells from the LL-OVAtreated group to naïve BALB/c mice demonstrated that these cells couldtransfer tolerance, as these cells were able to reduce the DTH responseafter immunizing and challenging the acceptor mice with OVA (FIG. 4).

Conclusion

Here, we demonstrated that intragastric administration of OVA-secretingL. lactis suppresses OVA-specific T cell responses via the induction ofCD4+ regulatory. We demonstrated that this immune tolerance induction ismore potent than free OVA protein, and that this could be established ina therapeutic setting.

Example B Induction of Antigen-Specific Oral Tolerance by GeneticallyModified Lactococcus lactis Delivering DQ8-Specific ImmunodominantGliadin Epitopes to Gluten-Sensitized Class II Transgenic MiceIntroduction

Celiac disease, also known as celiac sprue or gluten-sensitiveenteropathy, is a chronic inflammatory disease that develops from animmune response to specific dietary grains that contain gluten. Celiacis a complex multigenic disorder that is strongly associated with thegenes that encode the human leukocyte antigen variants HLA-DQ2 orHLA-DQ8. One of the most important aspects in the pathogenesis of Celiacis the activation of a T-helper 1 immune response. This arises whenantigen-presenting cells that express HLA-DQ2/DQ8 molecules present thetoxic gluten peptides to CD4(+) T-cells. Both classes of glutenproteins, gliadins and glutenins, contain peptides that bind DQ2 andDQ8. It is generally accepted that the immune response, such as theproduction of IFN-γ from gluten-specific T cells, triggers destructionof the mucosa in the small intestine of celiac disease patients. Hence,the activation of a detrimental immune T cell response in the intestineof celiac disease patients appears to be key in the initiation andprogression of the disease.

Antigen-specific immune suppression is an attractive therapeutic goalfor the treatment of celiac disease. Active delivery of recombinantgluten proteins/peptides at the intestinal mucosa by geneticallymodified Lactococcus lactis (LL) provides a novel therapeutic approachfor the induction of tolerance. For this purpose we geneticallyengineered deamidated DQ8 epitope secreting LL (LL-eDQ8d) and evaluatedthe local and systemic immune response in gluten-sensitized NOD AB^(o)DQ8 class II transgenic mice after oral supplementation.

Here, we demonstrate that oral delivery of gliadin peptide producing L.lactis suppresses gliadin-specific immune responses via the induction ofantigen-specific CD4⁺ regulatory T cells.

Materials and Methods

Bacteria and media: The Lactococcus lactis MG1363 (LL) strain wasgenetically modified and used throughout this study. Bacteria werecultured in GM17E medium, being M17 broth (Difco Laboratories, Detroit,Mich.) supplemented with 0.5% glucose and 5 μg/ml erythromycin (Abbott).Stock suspensions of LL strains were stored at −20° C. in 50% glycerolin GM17E medium. Stock suspensions were diluted 200-fold in GM17E mediumand incubated at 30° C. overnight. Within 16 h of culture, a saturationdensity of 2×10⁹ colony forming units (CFU) per ml was reached. Bacteriawere harvested by centrifugation and 10-fold concentrated in BM9inoculation buffer at 2×10⁹ bacteria/100 μl. For treatment, each mousereceived 100 μl of this suspension daily by intragastric catheter.

Plasmids: The sequence encoding the deamidated DQ8 epitope, (encodingDQ8d: caa tac cca tca ggt gaa ggt tca ttc caa cca tca caa gaa aac ccacaa gct (SEQ ID NO: 1)), was retrieved from published data. In summary,two glutamine residues within the alpha-gliadin peptide were changedinto glutamic acids to stimulate the deamidated immunodominantalpha-gliadin response for the DQ8 carrying celiac disease patients, andthis epitope is recognized by T cells of these mice. The DQ8d cDNAfragment was synthetically constructed (Operon, The Netherlands) andamplified by Polymerase Chain Reaction (PCR) using the following forwardand reverse primers 5′-caatacccatcaggtgaaggttc-3′ (SEQ ID NO: 11) and5′-cgactagttaagcttgtgggttttcttgtgat-3′ (SEQ ID NO: 12). For detectionpurposes an e-tag (e) was attached to the fragment, consisting of thefollowing sequence ggt gct cca gtt cca tac cca gat cca ctt gaa cca cgt(SEQ ID NO: 13). To add the e-tag to the 5′ end of DQ8d gene, the PCRproduct that was produced in step 1 (DQ8d) was used as template in a PCRwith oligonucleotides5′-ggtgctccagttccatacccagatccacttgaaccacgtcaatacccatca-3′ (SEQ ID NO:14) and 5′-cgactagttaagcttgtgggttttcttgtgat-3′ (SEQ ID NO: 15). Theamplified fragment was fused to the Usp45 secretion signal of theerythromycin resistant pT1 NX vector, downstream of the lactococcal P1promotor. MG1363 strains transformed with plasmids carrying eDQ8d cDNAwere designated Lactococcus lactis secreting eDQ8d (LL-eDQ8d). TheLL-pT1 NX, which is MG1363 containing the empty vector, pT1 NX, servedas control.

Functional analysis secreted epitopes: For functional analysis of thesecreted eDQ8d epitope a proliferation assay with human T cell clonesderived from the intestines of celiac disease (CD) patients wasperformed. Bacteria were grown overnight as described before, deluded1:50 and grown for another 4 or 6 hours respectively. T cell clonesspecific for gluten were generated from a small intestinal biopsy takenfrom patient S, an adult Dutch CD patient that had been on a gluten-freediet for several years. The patient gave informed consent to the study,which was approved by the hospital ethics committee. The patient wastyped serologically to be HLA-DR3/4, DQ2/8, thus carrying bothCD-associated DQ dimers. T cell clone 1129 was found to respond to analpha-gliadin derived peptide with a minimal 9 amino acid coreQGSFQPSQQ, when bound to HLA-DQ8. Deamidation of the P1 and/or P9glutamine residue (Q) into glutamic acid (E) by the activity of tissuetransglutaminase was found to substantially enhance the T cellstimulatory capacity of this gluten peptide. Proliferation assays wereperformed in duplicate or triplicate in 150 μl culture medium (Iscoves)in 96-well flat-bottomed plates (Falcon) using 10⁴ T cells stimulatedwith 10⁵ HLA-DQ-matched and 3000 RAD irradiated Peripheral bloodmononuclear cells in the absence or presence of supernatant at severalconcentrations. After 48 hours, cultures were pulsed with 0.5 uCi of³H-thymidine, harvested 18 hours thereafter upon which ³H-thymidineincorporation was determined as a measure for proliferation.

Mice: Transgenic mice that express HLA-DQ8 in an endogenous MHCII-deficient background (AB^(o) DQ8⁺) were backcrossed to NOD mice for10 generations and intercrossed to produce congenic NOD AB^(o) DQ8⁺mice. Seven to sixteen week old mice were used for the experiments. Micewere weaned and maintained in a conventional animal facility until 8-12weeks of age.

Antigen and Antibodies: Deamidated DQ8 epitopes with(GAPVPYPDPLEPRQYPSGEGSFQPSQENPQA (SEQ ID NO: 16)) and without(QYPSGEGSFQPSQENPQA (SEQ ID NO: 2)) e-tag were synthesized. For T-cellphenotyping, CD4 and CD25 antibodies were purchased from BD-Biosciences(San Jose, Calif.), and APC anti-Foxp3 staining kits were purchased fromeBiosciences (San Diego, USA) respectively. Anti-IL-10 neutralisingmonoclonal antibody (1 μg/ml, clone JES052A5), TGF-β neutralizingmonoclonal antibody (1 μg/ml, clone 1D11) and LAP neutralizingantibodies (1 μg/ml, clone 27235) were obtained from R&D systems(Minneapolis, Minn.).

Oral feeding and DTH (Delayed-type hypersensitivity) reaction: NODAB^(o) DQ8 mice on a gluten free chow were sensitized by subcutaneousinjection of 100 μg deamidated eDQ8 peptides in 100 μl of a 1:1 CFA(purchased from Difco of Becton, Dickinson and Company, San Jose,Calif.) saline solution in the tail base at day 1. The peptide used forthe sensitization had the same sequence as the secreted epitope. Micewere fed BM9 as a negative control, LL-pT1NX or LL-eDQ8d [all at days1-10 dissolved in 100 μl BM9]. Feedings were performed by intragastricadministrations of antigen or bacterial suspensions using an 18-gaugestainless gavage needle. Ten days after immunization, antigen-specificDTH responses were assessed. Twenty-four hours thereafter DTHmeasurements were performed. For measurement of antigen-specific DTHresponses, mice were challenged with 10 μg eDQ8d in 10 μl saline in theauricle of the ear. The increase in ear thickness was measured in ablinded fashion using an engineer's micrometer (Mitutoyo, Tokyo, Japan)at 24 h after challenge. DTH responses were expressed as the differencein increase 24 hours after eDQ8d injection, following subtraction ofear-thickness before challenge. Subsequently mice were sacrificed,spleen and lymph nodes were harvested and cells were assessed forDQ8d-specific proliferation and cytokine production. For e-taginterference NOD AB^(o) DQ8 mice were immunized with 100 μg deamidatedDQ8 peptides with (eDQ8d) or without E-tag (DQ8d) in 100 μl of a 1:1Complete Freund's Adjuvant (CFA, Difco, BD) saline solution in the tailbase at day 1. At day 7 mouse DTH measurements were performed asdescribed above with 10 μg DQ8d with or without e-tag, corresponding tothe peptide used for the immunization.

Cell cultures, proliferation and cytokine production assays: Cellsuspensions of spleen and lymph nodes were prepared at day 11 of theexperiment by homogenizing the tissue with a tissue grinder in 1×PBS.Erythrocytes were removed from the spleen cell suspensions by incubationwith ACK (Ammonium Chloride/Potassium (lysing buffer)). Cells wereincubated in 96-well microtiter plates at 5×10⁵ cells/well in 0.2-mlvolumes at 37° C. in RPMI 1640 (1.5% Hepes, 1% Penstrep and 10% FBS)with supplements containing either medium alone, 10 μg Con A, or 50 μgeDQ8d epitope. In a separate experiment IL-10, TGF-β, IL10&TGF-β or LAPneutralizing antibodies were added to splenocytes of LL-eDQ8d treatedmice. After 24 h, proliferation was assessed by addition of 1 μCi/well[³H]-thymidine for the last 24 h of culture. DNA-bound radioactivity washarvested onto glass fiber filter mats and thymidine-incorporationmeasured on a scintillation counter (Perkin Elmer). Results wereexpressed as mean cpm of triplicate wells. For cytokine measurements,supernatants of the cell cultures used in the different proliferationassays, described above, were collected after 24 h of culture and frozenat −20° C. until cytokine analysis was performed. Cytokine productionwas quantified using the Mouse Inflammation Cytometric Bead Assay (BDBiosciences).

Flow cytometric analysis: Spleens and gut-associated lymph node tissue(GALT) of BM9, LL-pT1 NX or LL-eDQ8d treated mice were isolated,prepared as described above and stained for CD4, CD25 and Foxp3.Intracellular staining was performed for Foxp3 according to themanufacturer's instructions (eBiosciences, San Diego, Calif.) andsubsequently measured using flow cytometry on a Becton DickinsonFACSCaliburs. For analysis cells were gated on CD4⁺CD25⁺ and CD4⁺CD25⁻subpopulations and within these populations Foxp3 histograms were usedto determine Mean Fluorescence Intensity (MFI).

Statistical analysis: Results from cytokine measurements are expressedas means±SEM. eDQ8d-specific proliferation, ear-thickness- and cytokinemeasurements were tested for significance using one-way ANOVA followedby the student's t-test comparison: two samples assuming equal variance,to determine the differences between individual groups. For all tests ap value <0.05: *, <0.01: ** was used to indicate statisticalsignificance for both tests.

Results

Mucosal Delivery of eDQ8d Epitopes by L. lactis Significantly Decreasesthe DQ8d-Induced DTH Response and Proliferative Capacity of Bulk Spleenand Inguinal Lymph Node Cells.

Daily intragastric administration of LL-eDQ8d in eDQ8d-immunized NODAB^(o) DQ8 class II transgenic mice led to a significant decrease in DTHresponse compared to the sensitized negative control mice (FIG. 5).Control mice (fed BM9) were clearly immunized to eDQ8d, but dailyintra-gastric administration of LL-eDQ8d significantly reduced the DTH(13.1×10⁻² mm vs 5.1×10⁻² mm, p=0.0031). Ear swelling was also slightlyreduced in LL-pT1NX-treated mice compared to controls (9.3×10⁻² mm vs13.1×10⁻² mm p=0.0343) but to a much lesser degree than in LL-eDQ8dtreated mice. Non DQ8 transgenic NOD AB^(o) mice showed only a minorincrease in ear thickness (3.2×10⁻² mm). These data indicate that orallyadministered LL-eDQ8d suppress systemic inflammatory T-cell responses inimmunized NOD AB^(o) DQ8 transgenic mice and that the secreted antigenis necessary for induction of a significant tolerogenic effect. Thesedata were accompanied by a significant decreased proliferative capacityof the splenocytes and inguinal lymph node cells (FIG. 6). The reducedproliferative response was accompanied by a significant up-regulation ofIL-10 and a downregulation of IL-12 production following ex vivo eDQ8dstimulation of splenocytes (FIG. 7). Moreover LL-eDQ8d significantlyreduced the eDQ8d-induced IFN-γ production in the inguinal lymph nodescompared to the BM9 and LL-pT1NX treated mice. Together, these dataindicate that LL-eDQ8d treatment suppresses T cell activation followingeDQ8d stimulation and suggest that DC activation my also be modulated.

Decreased Splenic eDQ8d-Specific Proliferation Depends on IL-10 andTGF-β, and LL-DQ8d Treatment Significantly Increases Splenic and GALTFoxp3 Expression

The functional importance of TGF-β, IL-10, and LAP (membrane-associatedTGF-β) on the eDQ8d-specific splenic proliferative response was analyzedusing neutralizing antibodies. IL-10-, TGF-β- or LAP-neutralizingantibodies did not significantly interfere with the decreased splenicproliferative response of LL-eDQ8d treated mice, but adding acombination of TGF-β and IL-10 neutralizing monoclonal antibodiescompletely abolished the decreased eDQ8d-specific proliferative capacityof splenocytes of LL-eDQ8d treated mice (FIG. 8). These data stronglysuggest that LL-eDQ8d treatment is able to suppress T cell activation ineDQ8d-immunized NOD AB^(o) DQ8 class II transgenic mice and that thissuppression is dependent on both IL-10 and TGF-β. Moreover, asignificant upregulation of Foxp3 was seen within the splenic CD4⁺CD25⁺as well as the CD4⁺CD25⁻ cell population of the LL-eDQ8d treated micecompared to the control (BM9) (MFI 171 vs 61 and 35 vs 6, respectively).Remarkably, Foxp3 was also upregulated in the CD4⁺CD25⁻ population inthe gut-associated lymph node tissue (GALT) of the LL-eDQ8d treated micecompared to the BM9 treated (MFI 73 vs 30), but not in the GALTCD4⁺CD25⁺ population. LL-pT1NX feeding also induced some Foxp3upregulation, but exclusively in the splenic CD4⁺CD25⁻ T-cell populationand to a lesser extent than LL-eDQ8d (MFI 15 vs 35, respectively).

Conclusion

Our data demonstrated that mucosal delivery of a gliadin derived peptideimmunodominant for DQ8 mediated T-cell responses-by genetically modifiedL. lactis, induces suppression of local and systemic DQ8 restrictedT-cell responses in NOD AB^(o) DQ8 class II transgenic mice. Treatmentresulted in an antigen-specific decrease of the proliferative capacityof the splenocytes and inguinal lymph node cells, which was criticallydependent on the production of IL-10 and TGF-β and associated with asignificant induction of Foxp3+ regulatory T-cells. Because thisapproach of antigen-delivering bacteria has the capacity forpotentiating oral tolerance even in the setting of establishedhypersensitivity, it may be applicable for the treatment of celiacdisease and possibly other autoimmune and/or allergic diseases.

Native DQ8 Epitope

The above experiments are repeated with the native α-gliadin epitope,i.e. QYPSGQGSFQPSQQNPQA (SEQ ID NO: 4), corresponding to residues203-220 of the sequence retrievable via UniProtKB/TrEMBL entry Q9M4L6.Said native DQ8 epitope is encoded by the nucleotide sequence 5′-caa taccca tca ggt caa ggt tca ttc caa cca tca caa caa aac cca caa get-3′ (SEQID NO: 3).

The results with the native α-gliadin DQ8 epitope are essentiallyidentical to the results described above for the deamidated α-gliadinDQ8 epitope.

Trial in Celiac Patients Using DQ8 Epitope

In a preliminary study, engineered L. lactis according to the inventionare used as a therapeutic in a trial in patients with Celiac disease.Our findings provide promise that this approach is effective in anantigen-specific manner.

Celiac disease is an especially attractive target for this approach, duetot the ability of the LL to deliver the antigen at the site of theprimary response to achieve both direct and bystander tolerance.

Trial in Celiac Patients Using DQ2 Epitope

No transgenic mice exist expressing HLA-DQ2 in an endogenous MHCII-deficient background, comparable to HLA-DQ8 mice as used above.Accordingly, the experiments described above for DQ8 epitopes were notpossible in an appropriate mouse model. We therefore conduct somepreliminary experiments in patients with celiac disease, using bothnative as well as deamidated α-gliadin DQ2 epitope.

Specifically, the above experiments are repeated using:

deamidated DQ2 epitope LQLQPFPQPELPYPQPQLPYPQPELPYPQPQPF (SEQ ID NO: 6),encoded by the nucleotide sequence 5′-tta caa tta caa cca ttc cca caacca gaa tta cca tac cca tta cca tac cca caa cca gaa tta cca tac cca caacca caa cca ttc (SEQ ID NO: 5)and the native DQ2 epitope: LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF (SEQ IDNO: 8), encoded by the nucleotide sequence 5′-tta caa tta caa cca ttccca caa cca caa tta cca tac cca tta cca tac cca caa cca caa tta cca taccca caa cca caa cca ttc (SEQ ID NO: 7)

The results with the native and deamidated α-gliadin DQ2 epitope areessentially identical to the results described above for the α-gliadinDQ8 epitopes.

Example C Induction of Tolerance to Clotting Factor VIII and Factor IXFollowing Oral Administration of L. lactis Secreting Said FactorsIntroduction

Several therapeutic (recombinant) proteins, such as interferon's, factorVIII/IX and antibodies (Remicade) are administered at high doses overprolonged treatment periods. However, a complication associated withtheir use is the development of protein-specific immune responses, suchas antibodies. These antibodies (Abs), also called inhibitors, renderthe therapeutic proteins less effective. Examples include the formationof inhibitors for factor VIII/IX in hemophilia, erythropoietin (Epo) inpatients undergoing therapy for chronic renal failure, and IFN- inpatients undergoing treatment for multiple sclerosis. Here, wedemonstrate that oral delivery of the Factor VIII (and Factor IX) by L.lactis suppresses inhibitor formation to said factor via the inductionof antigen-specific CD4⁺ regulatory T cells.

Material and Methods

Bacteria and plasmids: The L. lactis strain MG1363 is used throughoutthis study. Bacteria are cultured in GM17 medium, i.e. M17 (DifcoLaboratories, Detroit, Mich.) supplemented with 0.5% glucose. Stocksuspensions of all strains are stored at −20° C. in 50% glycerol inGM17. For intragastric inoculations, stock suspensions are diluted200-fold in fresh GM17 and are incubated at 30° C. They reach asaturation density of 2×10⁹ colony-forming units (CFU) per ml within 16hours. Throughout this study, mixed bacterial suspensions are used.Therefore, the bacteria that are mixed are harvested by centrifugationand pellets of both bacterial cultures are concentrated 10-fold in BM9medium (Schotte, Steidler et al. 2000). For treatment, each mousereceives 100 μl of this suspension by intragastric catheter.

Human FVIII and FIX cDNA or cDNA-fragments, representing FVIII- andFIX-specific CD4+ T-cell epitopes, are amplified fused to the Usp45secretion signal of the erythromycin resistant pT1 NX vector, downstreamof the lactococcal P1 promotor.

MG1363 strains transformed with plasmids carrying human FVIII (and/orepitope fragment), FIX (and/or epitope fragment), were designated L.lactis secreting LL-FVIII, LL-FIX. LL-pT1 NX, which is MG1363 containingthe empty vector pT1 NX, serve as control.

Quantification of FVIII and FIX: FVIII or FIX from LL-FVIII and LL-IX,respectively are determined using human FVIII and FIX-specificenzyme-linked immunosorbent assay (ELISA), that have been describedpreviously (Chuah et al., 2003). The recombinant proteins are alsoanalyzed by Western blot analysis and COATests and aPTT assays, asdescribed (Chuah et al., 2003; VandenDriessche et al., 1999). TheNH2-terminus of this protein is determined by automated Edmandegradation. Since FVIII and FIX are normally expressed in the liverwhere they undergo extensive post-translational modifications, theclotting factors produced from the engineered L. lactis may bebiologically inactive. However, these post-translational differenceswill likely have no repercussions on the ability of these L.lactis-produced recombinant proteins to induce immune tolerance. Indeed,most inhibitors that have been characterized in detail to date typicallyrecognize amino acid residues (Villard et al., 2003), rather thanglycosylated moieties.

Animals: Hemophilia A or B mice obtained by knocking-out the murineFVIII or FIX genes using homologous recombination in ES cells asdescribed by (Bi et al., (1995) and Wang et al., (1997), are bred in thelaboratory. These recipient mice generate neutralizing antibodies whenchallenged with purified recombinant FVIII or FIX antigen in thepresence of CFA (Mingozzi et al., 2003). The inhibitor status can bemonitored over time using Bethesda assays or anti-FVIII/anti-FIXspecific ELISAs. Recipient mice challenged with FVIII or FIX (+CFA)typically develop inhibitors 2-3 weeks after antigenic challenge.

Experimental setting: 4-6 week-old mice receive FVIII, FIX, LL-FVIII,LL-FIX, or LL-pT1NX or LL-OVA (an irrelevant antigen) (1 or 10 μg). As apositive control for tolerance induction, we inject mice withadeno-associated viral vectors (AAV) expressing FIX from ahepatocyte-specific promoter. Recipient animals develop FIX-specificimmune tolerance that prevents induction of anti-FIX antibodies uponsubsequent challenge with FIX+CFA.

In a prophylactic setting, FVIII, FIX, LL-FVIII, LL-FIX alone areadministered orally to hemophilia A or B mice using a gastric catheter,using different treatment intervals and doses. These recipient mice aresubsequently challenged with purified recombinant FVIII or FIX antigen,in the presence of CFA (Mingozzi et al., 2003). Control animals areexposed to LL-pT1NX and LL-OVA. Plasma is harvested by retro-orbitalbleeding. The development of antibodies directed against FVIII or FIX isassessed using Bethesda assays (Kasper et al., 1975) or using a modifiedanti-FVIII or anti-FIX specific ELISA (VandenDriessche et al., 1999) atdifferent time intervals.

In a therapeutic setting, hemophilia A or B mice are first immunizedwith FVIII or FIX, as described (Mingozzi et al., 2003). The inhibitorstatus is monitored over time using Bethesda assays oranti-FVIII/anti-FIX specific ELISAs. Mice with low or high inhibitortiters are subsequently treated with FVIII, FIX, LL-FVIII, LL-FIX aloneusing different treatment intervals and doses and inhibitor titers aredetermined over time. The specificity of the possible immune toleranceis assessed by challenging the mice that receive FVIII, FIX, LL-FVIII,LL-FIX alone with an irrelevant antigen (tetanus toxoid or Ova).

Cell cultures, proliferation and cytokine assay: Single cell suspensionsof spleen and lymph nodes are prepared by passing the cells through 70μm filter cell strainers (Becton/Dickinson Labware). Erythrocytes areremoved from the spleen cell suspensions by incubation with red celllysis buffer.

Proliferation assays of total splenocyte populations, 2×10⁵ cells arecultured in 96-well U-bottom plates in a total volume of 200 μl completemedium either alone or with purified FVIII or FIX, and either with orwithout anti-IL-10 or anti-TGF-β neutralising monoclonal antibodies.FVIII and FIX is added at concentrations ranging from 1 to 100 μg/ml.The neutralizing antibodies are added at 1, 0.1 and 0.01 μg/ml. Forproliferation assays of CD4⁺ T cells and CD4⁺CD25⁻ T cell populations,0.2×10⁵ cells CD4⁺ T cells or CD4⁺CD25⁻ T cells are cultured in 96-wellU-bottom plates with 1×10⁵ irradiated CD4⁻ cells, acting as antigenpresenting cells, and FVIII or FIX (0 or 100 μg/ml) in a total volume of200 μl complete medium either with or without neutralizing antibodies.After 72 hr at 37° C. in a 5% CO₂ humidified incubator, proliferation isassessed by addition of 1 μCi/well [³H]-thymidin. DNA-boundradioactivity is harvested 16-18 hr later onto glass fiber filter mats(Perkin Elmer, Boston, USA) and thymidine-incorporation is measured on ascintillation counter (Perkin Elmer).

For cytokine measurements, supernatants of the cell cultures used in thedifferent proliferation assays are collected after 24, 48 and 72 h ofculture and frozen at ±20° C. until cytokine analysis is performed.Cytokine production is quantified using the Mouse InflammationCytometric Bead Assay (BD Biosciences, Mountain View, Calif., USA).

In vivo T regulatory activity assay: In order to test for activesuppression of antibody formation in mice, splenocytes, bead-purifiedCD4⁺ T cells, CD4⁺CD25⁻ or CD4⁺CD25⁺ T cells isolated from the differentexperimental L. Lactis-treated groups are adoptively transferred tonaïve C3H/HeJ mice. Untreated mice are used as control. The number oftransferred cells is 10⁷ for whole spleen cells, subpopulation-depletedspleen cells, or positively selected CD4⁺ cells and CD4⁺CD25⁻ andCD4⁺CD25⁺ T cells. Recipient mice (n=4-5 per experimental cohort) weresubcutaneously injected with 5 μg hF.IX in cFA 36 hours after adoptivetransfer. Anti-hF.IX IgG titers in plasma were measured 2.5 weeks afterimmunization.

Results LL-FVIII and LL-IX Significantly Enhances the Tolerance-InducingCapacity of in Hemophilia A or B Mice Compared to Free FVIII or FIX

To study the induction of oral tolerance, mice are orally fed asdescribed above (experimental setting). Addition of LL-FVIII or LL-FIXsignificantly enhances the tolerance induction towards FVIII and FIX asthe factor-specific proliferative response of splenocytes issignificantly reduced in this group in comparison to the control andfree FVIII and FIX groups.

LL-FIIIV and LL-FIX Potentiate Oral Tolerance in Association withReduced FVIII- and FIX-Specific Titers and IFN-γ and More IL-10 andTGF-β Production in Response to Said Factor.

To study the induction of oral tolerance, mice are orally fed asdescribed above (experimental setting). FVIII and FIX-specificantibodies and cytokine production in response to said factor insplenocytes and lymph nodes are quantified as described above. Theinhibitor formation and production of the proinflammatory cytokine,IFN-γ is strongly reduced and the immunosuppressive cytokines IL-10 andTGF-β is significantly increased in the LL-FVIII/FIX group in comparisonto the control and free FVIII/IX groups.

LL-FVIII/FIX Enhances Oral Tolerance Via CD4+ T Cells

To assess whether CD4+ T cells mediate the induction of oral tolerance,the factor-specific proliferative CD4+ T-cell response is studied in thesplenocytes and lymph nodes. Therefore, mice are orally fed as describedabove (experimental setting) and the factor-specific CD4+ T cellproliferation is determined as described in Cell cultures, proliferationand cytokine assay. The factor-specific CD4 T-cell response in theLL-FVIII/FIX group is significantly reduced in comparison to the controland free FVIII/IX groups.

Antigen-Induced T Regulatory Cells Following LL-FVIII/FIX Therapy CanTransfer Protection From Inhibitor Formation In Vivo

In order to test for active suppression of antibody formation in micetreated with the oral tolerance protocol, we adoptively transfersplenocytes from the different treated groups as described above (Invivo T regulatory activity assay). Compared with controls and freeFVIII/IX groups, antifactor IgG formation is significantly reduced inthe LL-FVIII/FIX group, indicating activation of regulatory CD4⁺ T cellsin our combination oral tolerance protocol.

Conclusion

Our data demonstrate that mucosal delivery of recombinant FVIII- or FIXsecreting L. lactis are more potent than free FVIII or FIX insuppressing the formation of FVIII- and FIX-specific inhibitors inHemophilia A and B mice respectively.

Example D Induction of Tolerance to an Allergen, Der p 1 Following OralAdministration of L. lactis Secreting Said Allergen Introduction

Allergic asthma is a chronic inflammatory disorder of the airways. It ischaracterized by reversible airway obstruction, elevated serum levels ofallergen-specific immunoglobulin E, mucus hypersecretion and airwayhyperresponsiveness (AHR) to ronchospasmogenic stimuli. Its symptoms aremade worse by exposure to an allergen (e.g., tree, grass and weedpollen, dust and dust mites, mold, animal dander) to which the patienthas been sensitized. Type 2 T-helper (Th2) lymphocytes play a crucialrole in the initiation, progression and persistence of the disease.Current data suggest that Th2 responses to allergens are normallysuppressed by regulatory T cells. Furthermore, suppression by thissubset is decreased in allergic individuals. Here, we demonstrate thatoral delivery of allergen by L. lactis suppresses asthma-like responsesvia the induction of antigen-specific CD4⁺ regulatory T cells.

Material and Methods

Two Mouse models of allergic asthma that mimics human disease are theOva allergen model and the humanized SCID model.

The Ova allergen model: OVA-sensitized mice are inhalationallychallenged with OVA aerosol that leads to Th2 cytokine-dependenteosinophilic airway inflammation, bronchial hyperreactivity, and IgEproduction, findings highly characteristic of human allergic asthma(Brusselle, 1994, Clin Exp Allergy 24:73; Kips et al. 1996, Am J RespirCrit. Care Med 153:535; Brusselle et al. 1995, Am J Respir Cell Mol Biol12:254).

Bacteria: The L. lactis strain MG1363 is used throughout this study.Bacteria are cultured in GM17 medium, i.e. M17 (Difco Laboratories,Detroit, Mich.) supplemented with 0.5% glucose. Stock suspensions of allstrains are stored at −20° C. in 50% glycerol in GM17. For intragastricinoculations, stock suspensions are diluted 500-fold in fresh GM17 andincubated at 30° C. They reached a saturation density of 2×10⁹colony-forming units (CFU) per mL within 16 hours. Bacteria areharvested by centrifugation and concentrated 10-fold in BM9 medium. Fortreatment, each mouse receives 100 μL of this suspension daily byintragastric catheter.

Plasmids: The mRNA sequence encoding Gallus gallus Ovalbumin isretrieved from Genbank (accession number AY223553). Total RNA isisolated from chicken uterus and cDNA is synthesized using 2 μg totalRNA, 2 μM oligo dT primers (Promega Corporation Benelux, Leiden, TheNetherlands), 0.01 mM DTT (Sigma-Aldrich, Zwijndrecht, The Netherlands),0.5 mM dNTP (Invitrogen, Merelbeke, Belgium), 20 U Rnasin (PromegaIncorporation Benelux) and 100 U superscript II reverse transcriptase(Invitrogen) in a volume of 25 μl. OVA cDNA fragment is amplified byPolymerase Chain Reaction (PCR) using the following conditions: 94° C.for 2 min followed by 30 cycles at 94° C. for 45 seconds, 62° C. for 30seconds and 72° C. for 90 seconds, with the following forward andreverse primers 5′-GGCTCCATCGGTGCAGCAAGCATGGAATT-3′ (SEQ ID NO: 17) and5′-ACTAGTTAAGGGGAAAC-ACATCTGCCAAAGAAGAGAA-3′ (SEQ ID NO: 18).

The amplified fragment is fused to the Usp45 secretion signal of theerythromycin resistant pT1 NX vector, downstream of the lactococcal P1promotor.

MG1363 strains transformed with plasmids carrying OVA cDNA aredesignated LL-OVA. LL-pTREX1, which is MG1363 containing the emptyvector, serve as control.

Quantification of OVA: OVA from LL-OVA are determined using an in housedeveloped OVA-specific enzyme-linked immunosorbent assay (ELISA).Production of the recombinant proteins is also assessed by Western blotanalysis.

Mice: BALB/c mice (6 to 8 weeks of age) are purchased from Charles RiverLaboratories (Calco, Italy). The mice are maintained under specificpathogen-free conditions.

Immunization of mice: Mice are immunized i.p. with 10 μg of OVA (gradeV; Sigma-Aldrich) in 1 mg of aluminum hydroxide (alum). Thisimmunization is repeated after 21 days (on days 0 and 21). Control micereceive a saline injection instead of the OVA/alum solution. 26 daysafter the immunization, sensitized mice inhale an aerosolized solutionof 1% OVA dissolved in PBS for 10 min. OVA inhalation is conducted for 3days in a row (days 47, 48, and 49). Control mice inhale PBS alone underthe same conditions as used for the experimental group.

Induction of oral tolerance: Mice receive LL-OVA, LL-pTREX1, 1 μg OVA orBM9 on days 0-4, 7-11, 14-18 and 21-25. As positive control for oraltolerance induction mice are fed 30 mg OVA by intragastric catheter thatreduce bronchial eosinophilia and airway hyperresponsiveness, with highdose feeding being more effective than low-dose feeding.

Measurement of airway hyperresponsiveness (AHR): 24 h after the finalinhalation (day 50), airway hyperresponsiveness is assessed bymethacholine-induced airflow obstruction. The mice are exposed for 2.5min to nebulized physiologic saline (Otsuka Pharmaceutical), followed byincremental doses (1-30 mg/ml) of nebulized methacholine. These mice areplaced in a whole-body plethysmograph for 2.5 min followingnebulization, and enhanced pause (Penh) is measured using Biosystem XAWBP system (Buxco Electronics). “Penh” represents pulmonary airflowobstruction and is calculated using the formula:Penh=((Te−Tr)/(Tr×PEF/PIF)), where Penh=enhanced pause (dimensionless),Te=expiratory time (seconds), Tr=relaxation time (seconds), PEF=peakexpiratory flow (milliliters per second), and PIF=peak inspiratory flow(milliliters per second). Penh is measured and averaged approximatelyevery 5 s, and the cumulative values are averaged as the Penh value foreach time point. Airway hyperresponsiveness is expressed as PC200Mch(200% provocative concentration of methacholine), which is theconcentration of methacholine that doubled the baseline Penh value.

Analysis of bronchoalveolar lavage fluid (BALF): After the measurementof airway hyperresponsiveness, bronchoalveolar lavage samples areobtained. The mice are euthanised by i.p. injection of overdose ketaminand xylazin, and then the lungs are lavaged with 0.5 ml of saline fourtimes. The lavage fluid is centrifuged and the cells are resuspended in1 ml of saline with 1% BSA. Total cell numbers are counted using ahemocytometer. Cytospin samples are prepared by centrifuging thesuspensions at 300 rpm for 5 min. To clearly distinguish the eosinophilsfrom the neutrophils, three different stains are applied: Diff-Quick,May-Grünwald-Giemsa, and Hansel (eosin) stains. At least 300 leukocytesare differentiated by light microscopy based on the standard morphologiccriteria. The level of IL-13, IL-4 and IL-5 in BALF is detected byCytometric Bead Assay (BD Biosciences, Mountain View, Calif., USA)following the manufacturer's instructions.

Measurement of serum total IgE and OVA-specific Ig: On day 50, bloodsamples are obtained from retro-orbital sinus under anesthesia. Afterthe samples had fully coagulated, they are centrifuged, and the sera iscollected and stored at −80° C. until use. Total IgE is assayed by ELISAusing paired Abs (BD Pharmingen) according to the manufacturer'sinstructions. To measure OVA-specific IgE, IgG1, and IgG2a in sera,microtiter plates (Maxisorp, Nunc, VWR International, Haasrode, Belgium)are coated with 2 μg/ml OVA. Subsequently, the wells are blocked with0.1% casein in PBS, after which the plates are incubated with mouseserum samples diluted 1:10 to 1:20480 in PBS containing 0.1% casein and0.05% Tween 20 (PBS-CT), with goat anti-mouse IgG2a-HRP [SouthernBiotechnology Associates (SBA), Imtec ITK Diagnostics, Antwerpen,Belgium, dilution 1:5000], goat anti-mouse IgG1-HRP or goat anti-mouseIgE-HRP (SBA, dilution 1:5000). After washing, substrate [3,3′,5,5′tetramethylbenzidine (TMB) substrate reagent, Pharmingen, BectonDickinson, Erembodegem, Belgium] is added to each well. Finally,reactions are stopped by adding 1M H₂SO₄ to the wells. The absorbancesare read at 450 nm. ELISA scores are expressed as titers, which are theinverse of the highest dilution that still had on (OD₄₅₀ higher than thecalculated cutoff value. The cutoff is calculated as the mean OD₄₅₀ of 5non-immunized mice increased with three times the SD.

Histological examination of lung tissue: After bronchoalveolar lavagesamples are obtained, the lungs are perfused with physiologic saline andare resected from the mice. The lungs are fixed with neutralizedbuffered formalin and embedded in paraffin. Sections (3-μm thick) arestained with H&E or periodic acid-Schiff (PAS). The intensity ofhistological changes in the lungs is evaluated with four grading scores(0, no inflammation; 1, slight/mild; 2, moderate; and 3, severe),according to the distribution and intensity of the followingfindings: 1) epithelial shedding or undulation of the nuclei ofbronchial epithelial cells, 2) increase in the number of goblet cells,3) infiltration of inflammatory cells from vessels into the mucosal andsubmucosal area of the bronchus and peribronchial interstitium, and 4)hypertrophy and thickening of the smooth-muscle cell layer.

RT-PCR for analysis of cytokine and chemokine gene expression in thelung: The lungs are removed after perfusion with physiologic saline, andtotal RNA is extracted using ISOGEN (Nippon Gene) according to themanufacturer's instructions. Total RNA (10 μg) is reverse-transcribedusing oligo(dT)15 primer (Promega) and Superscript II RNase H-reversetranscriptase (Invitrogen Life Technologies) at 42° C. for 2 h. Toensure that each sample contained the same amount of cDNA, the β-actincDNA concentration of each sample is first determined usingβ-actin-specific primers. These samples are amplified for theappropriate number of cycles, such that the amount of PCR productremained on the linear part of the amplification curve. The PCR productsare electrophoresed in a 2% agarose gel and were visualized by ethidiumbromide staining. The levels of IL-13, eotaxin, IL-10, IFN-γ, and TGF-βare determined using the following specific primer sets.

The sense primer for β-actin 5′-ACGACATGGAGAAGATCTGG-3′, (SEQ ID NO: 19)and the antisense primer 5′-TCGTAGATGGGCACAGTGTG-3′. (SEQ ID NO: 20) Thesense primer for IL-13 5′-TCTTGCTTGCCTTGGTGGTCTCGC-3′, (SEQ ID NO: 21)and the antisense 5′-GATGGCATTGCAATTGGAGATGTTG-3′. (SEQ ID NO: 22) Thesense primer for eotaxin 5′-GGGCAGTAACTTCCATCTGTCTCC-3′, (SEQ ID NO: 23)and the antisense primer 5′-CACTTCTTCTTGGGGTCAGC-3′. (SEQ ID NO: 24) Thesense primer for IL-10 5′-TACCTGGTAGGAGTGATGCC-3′, (SEQ ID NO: 25) andthe antisense 5′-GCATAGAAGCATACATGATG-3′. (SEQ ID NO: 26) The senseprimer for IFN-γ 5′-CATAGATGTGGAAGAAAAGA-3′, (SEQ ID NO: 27) and theantisense 5′-TTGCTGAAGAAGGTAGTAAT-3′. (SEQ ID NO: 28) The sense primerfor TGF-β 5′-CTTTAGGAAGGACCTGGGTT-3′, (SEQ ID NO: 29) and the antisense5′-CAGGAGCGCACAATCATGTT-3′. (SEQ ID NO: 30)

Cell cultures, proliferation and cytokine assay: One day after the finalinhalation (day 50) single cell suspensions of spleen and mediastinallymph nodes are prepared by passing the cells through 70 μm filter cellstrainers (Becton/Dickinson Labware). Erythrocytes are removed from thespleen cell suspensions by incubation with red cell lysis buffer. CD4⁺ Tcells and CD4⁺CD25⁻ T cells are enriched using CD4⁺ T cell isolation kit(Miltenyi Biotec, Germany) or CD4⁺CD25⁺ regulatory T cell isolation kit(Miltenyi Biotec, Germany), respectively and MACS columns (midiMACS;Miltenyi Biotec).

Proliferation assays of bulk splenocyte and LN populations, 2×10⁵ cellsare cultured in 96-well U-bottom plates in a total volume of 200 μlcomplete medium either alone or with purified OVA. OVA is added atconcentrations ranging from 1 to 100 μg/ml. For proliferation assays ofCD4⁺ T cells and CD4⁺CD25⁻ T cell populations, 2×10⁵ cells CD4⁺ T cellsor CD4⁺CD25⁻ T cells are cultured in 96-well U-bottom plates withmitomycin treated splenocytes that are loaded with 1 mg/ml OVA for 16 h,acting as antigen presenting cells, at ratio's CD4⁺ T cell or CD4⁺CD25⁻T cell/APCs 1/1, 1/0.3, 1/0.1, 1/0.03, 1/0 in a total volume of 200 μlcomplete medium. After 72 h at 37° C. in a 5% CO₂ humidified incubator,proliferation is assessed by addition of 1 μCi/well [³H]-thymidin.DNA-bound radioactivity is harvested 18 h later onto glass fiber filtermats (Perkin Elmer, Boston, USA) and thymidine-incorporation is measuredon a scintillation counter (Perkin Elmer).

For cytokine measurements, supernatants of the cell cultures used in thedifferent proliferation assays is collected after 24, 48 and 72 h ofculture and frozen at −80° C. until cytokine analysis is performed.Cytokine production is quantified using the Mouse InflammationCytometric Bead Array (BD Biosciences, Mountain View, Calif., USA).

In vivo T regulatory activity assay: One day after the final inhalation(day 21), spleens of the treated mice are digested with 0.1% collagenase(Sigma-Aldrich) at 37° C. for 20 min. In some experiments, single-cellsuspensions of whole spleen cells are prepared and cultured with Con A(2 μg/ml; Sigma-Aldrich) for 48 h. Cells are collected, and 10⁷ cellsare adoptively transferred i.v. into naïve BALB/c mice. For negativeselection, CD4⁺, CD8⁺, CD11c⁺, CD19⁺, or CD11b⁺ cells are depleted fromthe whole spleen cells using magnetic beads (MACS; Miltenyi Biotec) withbiotinylated anti-mouse CD4, CD8, CD11c, CD19, and CD11b mAb (BDPharmingen), according to the manufacturer's instructions. Theefficiency of depletion is examined by flow cytometry (>99%). CD4⁺,CD4⁺CD25⁻ cells are purified using CD4⁺ T cell isolation kit. RegulatoryT cell isolation kit following the manufacturer's instructions. Thepurity of positively selected cells is checked using flow cytometry. Forcell transfer experiments, cells are transferred into BALB/c mice fromthe tail veins just before their first immunization or just after theirsecond immunization with OVA/alum. The number of transferred cells is10⁷ for whole spleen cells, subpopulation-depleted spleen cells, orpositively selected CD4⁺ cells and CD4⁺CD25⁻ cells.

In the Humanized SCID (hu-SCID) Model (as Described by Duez et al.,2000; Hammad et al., 2000)

In this model, the allergic immune response to the house dust mite (HDM)allergen Der p 1 can be studied. Such hu-SCID mice reconstituted i.p.with PBMC from HDM-allergic patients and subsequently exposed toaerosols of HDM produce human IgE, develop a pulmonary infiltratecomposed of activated T cells and DCs, and exhibit AHR in response tobronchoconstrictor agents (Pestel et al. 1994, J Immunol, 153:3804; Duezet al., Am J Respir Crit. Care Med, vol 161, ppp 200-206, 2000).

Bacteria

The L. lactis strain MG1363 is used throughout this study. Bacteria arecultured in GM17 medium, i.e. M17 (Difco Laboratories, Detroit, Mich.)supplemented with 0.5% glucose. Stock suspensions of all strains arestored at ±20° C. in 50% glycerol in GM17. For intragastricinoculations, stock suspensions are diluted 200-fold in fresh GM17 andincubated at 30° C. They reached a saturation density of 2×10⁹colony-forming units (CFU) per mL within 16 hours. Bacteria areharvested by centrifugation and concentrated 10-fold in BM9 medium. Fortreatment, each mouse receives 100 μL of this suspension daily byintragastric catheter.

Plasmids

Der p 1, a 222 amino-acid residue globular glycoprotein, is one of themajor allergens from Dermatophagoides pteronyssinus (Dpt) mites. DNAsequence with optimal L. lactis codon usage encoding the Der p 1 proteinis synthesized, amplified and fused to the Usp45 secretion signal of theerythromycin resistant pT1 NX vector downstream of the lactococcal P1promotor. MG1363 strains transformed with plasmids carrying murine Der p1, Der p 1 aa52-71 and Der p 1 aa117-133 cDNA are designated LL-Derp1,LL-Derp1aa52-71 and LL-Derp1aa117-133. LL-pT1 NX, which is MG1363containing the empty vector pT1 NX, serve as control.

Quantification of Der p 1

Der p 1 from LL-Derp1 is determined using an in house developed Der p1-specific enzyme-linked immunosorbent assay (ELISA). Production of therecombinant proteins is also assessed by Western blot analysis.

Patients

Blood is collected from donors sensitive or not sensitive to house dustmites. Allergic patients present the usual features of house dust mitesensitization. Skin prick tests toward Dermatophagoides pteronyssinus(Dpt) allergen (Stallergènes, Fresnes, France)(diameter≧10 mm) arepositive, and all patients have serum specific IgE antibodies. Total IgEconcentrations are greater than 150 IU/ml (150-1600 IU/ml). Healthydonors are tested as negative controls (total IgE levels are less than150 IU/ml, and they have negative skin prick tests toward commonlyinhaled allergens).

Human Peripheral Blood Mononuclear Cell Preparation

Platelet rich plasma is obtained after centrifugation (120×g, 15minutes) and discarded. Blood cells are then diluted in RPMI 1640 (LifeTechnologies, Paisley, Scotland) (vol/vol) and layered over a Ficollgradient (Pharmacia, Uppsala, Sweden). After centrifugation (400×g, 30minutes), PBMCs are harvested at the interface and washed three times insterile RPMI medium before transfer.

Mice

C.B.-17 SCID mice (6-8 weeks old) are maintained in isolators withsterilized bedding in a specific animal facility. The SCID colony isregularly checked for absence of mouse serum immunoglobulins by ELISA.

Peripheral Blood Mononuclear Cells Transfer in SCID Mice: PBMC hu-SCIDMice

SCID mice are between 6 and 8 weeks old at the time of cell transfer.The mice are reconstituted by intraperitoneal injection of 10×10⁶mononuclear cells from allergic patients or healthy donors in 400 μl ofRPMI via a 23-gauge needle. On the same day, they receiveintraperitoneally 2 index reactivity [IR] units Dpt. Four days after thecell reconstitution, SCID mice are exposed to daily allergen aerosolscontaining 100 IR units of Dpt (100 IR units are equivalent toapproximately 200 μg of protein contained in the Dpt extract) for 4successive days (day 0 to day 4). The control group is not exposed toDpt. One day before airway responsiveness measurement (day 35 and day60), hu-SCID mice are exposed to another aerosol of 100 IR units of Dptsolution.

Experimental Setting

Mice receive L. lactis engineered to express Der p 1 or an irrelevantantigen (OVA) as negative control.

The engineered L. lactis bacteria are administered orally to SCID miceusing a gastric catheter, using different treatment intervals and dosesstarting one day after PBMC reconstitution. Induction of oral toleranceis assessed by measuring human serum IgE antibodies, analysis ofpulmonary infiltration, measurement of AHR and analysis of cellpopulations and cytokine production in the BALF. Furthermore, inductionof tolerance is assessed by analysis of the proliferative T cellresponse against Der p 1.

Assessment of Airway Responsiveness (AHR)

Airway responsiveness (expressed as provocative dose of carbacholcausing a 50% increase in lung resistance) is measured on day 35 or day60 as described by Duez et al. 2000.

Human IgE Measurements

Several days after transplantation with human cells, mice are bled fromthe retro-orbital sinus under anesthesia. Total human IgE isinvestigated by a two-site immuno-radiometric method with the use of twodifferent mouse mAbs specific for the E-chain (Immunotech International,Luminy, France). At least 20 μl of serum is used in a duplicate test.The sensitivity of the method permits the detection of 0.1 IU/ml (0.24ng/ml).

Specific IgE Ab against Dpt allergen is quantified by ELISA. Briefly,plastic tubes (Maxisorb Startube, Nunc, Denmark) are coated overnightwith Dpt allergen in 0.1 M carbonate/bicarbonate buffer (pH 9.6) at 4°C. and saturated with 1% BSA in 0.1 M PBS (pH 7.4) for 2 h at roomtemperature. After washing, the tubes are incubated for 2 h at roomtemperature and overnight at 4° C. with Hu-SCID mice serum diluted inPBS containing BSA (1%) and Tween (0.01%). After extensive washings, aHRP-labeled anti-human IgE Ab is added. After washing, substrate[3,3′,5,5′ tetramethylbenzidine (TMB) substrate reagent, Pharmingen,Becton Dickinson, Erembodegem, Belgium] is added to each well. Finally,reactions are stopped by adding 1M H₂SO₄ to the wells. The absorbancesare read at 450 nm.

Histological Examination of the Lung

Lungs are excised at day 35 and fixed in paraformaldehyde and processedfrom paraffin embedding. Paraffin tissue sections are stained for thedetection of human CD45+ cells after which human cells on the murinelung sections were quantified by histological scoring as described byDuez et al. 2000.

Analysis of Bronchoalveolar Lavage Fluid (BALF)

BALF is analyzed as described in the OVA allergen model.

Cell Cultures, Proliferation and Cytokine Assay:

Single cell suspensions of spleen are prepared by passing the cellsthrough 70 μm filter cell strainers (Becton/Dickinson Labware).Erythrocytes are removed from the spleen cell suspensions by incubationwith red cell lysis buffer. CD4⁺ T cells and CD4⁺CD25⁻ T cells areenriched using human CD4⁺ T cell isolation kit (Miltenyi Biotec,Germany) or human CD4⁺CD25⁺ Regulatory T cell isolation kit (MiltenyiBiotec, Germany), respectively and MACS columns (midiMACS; MiltenyiBiotec).

Proliferation assays of bulk splenocyte, 2×10⁵ cells are cultured in96-well U-bottom plates in a total volume of 200 μl complete mediumeither alone or with purified Der p 1, and either with or withoutanti-IL-10 or anti-TGF-β neutralising monoclonal antibodies. Der p 1 isadded at concentrations ranging from 1 to 100 μg/ml. The neutralizingantibodies are added at 1, 0.1 and 0.01 μg/ml. For proliferation assaysof human CD4⁺ T cells and human CD4⁺CD25⁻ T cell populations, 2×10⁵cells CD4⁺ T cells or CD4⁺CD25⁻ T cells are cultured in 96-well U-bottomplates with mitomycin treated human PBMC that are loaded with 1 mg/mlDer p 1 for 16 h, acting as antigen presenting cells, at ratio's CD4⁺ Tcell or CD4⁺CD25⁻ T cell/APCs 1/1, 1/0.3, 1/0.1, 1/0.03, 1/0 in a totalvolume of 200 μl complete medium either with or without neutralizingantibodies. After 72 h at 37° C. in a 5% CO₂ humidified incubator,proliferation is assessed by addition of 1 μCi/well [³H]-thymidin.DNA-bound radioactivity is harvested 18 h later onto glass fiber filtermats (Perkin Elmer, Boston, USA) and thymidine-incorporation is measuredon a scintillation counter (Perkin Elmer).

For cytokine measurements, supernatants of the cell cultures used in thedifferent proliferation assays is collected after 24, 48 and 72 h ofculture and frozen at −80° C. until cytokine analysis is performed.Cytokine production is quantified using the Human InflammationCytometric Bead Assay (BD Biosciences, Mountain View, Calif., USA).

Results

LL-OVA and LL-Der p 1 Significantly Enhances the Tolerance-InducingCapacity in OVA- and huSCID Mice Model For Asthma, Respectively.

To study the induction of oral tolerance, mice are orally fed asdescribed above (experimental setting). Addition of LL-OVA/Derp1significantly enhances the tolerance induction towards OVA/Derp1 as theallergen-specific proliferative response of the splenocytes issignificantly reduced in the LL-OVA/Derp1 group in comparison to thecontrol and free OVA/Derp1 groups.

LL-OVA/Derp1 Potentiates Oral Tolerance in Association with Reduced AHR,Eosinophilic Infiltration, Serum IgE Levels, and Lowered IL-13, IL-4 andIL-5 Cytokine Production in Response to Said Allergen.

To study the induction of oral tolerance, mice are orally fed asdescribed above (experimental setting). AHR, eosinophilic BALFinfiltration, IgE titer as well as cytokine production in response tosaid antigens is determined as described above. AHR, eosinophilic BALFinfiltration, IgE titer is strongly reduced, and IL-13, IL-4 and IL-5significantly lowered in the LL-OVA/Derp1 group in comparison to thecontrol and free OVA/Derp1 groups.

LL-OVA/Derp1 Enhances Oral Tolerance Via CD4+ T Cells.

To assess whether CD4 T cells mediate the induction of oral tolerance,the allergen-specific proliferative CD4 T-cell response is studied inthe splenocytes and lymph nodes. Therefore, mice are orally fed asdescribed above (experimental setting) and the allergen-specific CD4+ Tcell proliferation is determined as described in Cell cultures,proliferation and cytokine assay. The allergen-specific CD4 T cellresponse in the LL-OVA/Derp1 group is significantly reduced incomparison to the control and free-OVA/Derp1 groups.

Antigen-Induced T Regulatory Cells Following LL-OVA Therapy can TransferProtection from Asthma-Like Responses In Vivo

In order to test for active suppression of asthma-like responses in micetreated with the oral tolerance protocol, we adoptively transfersplenocytes from the different treated groups as described above (Invivo T regulatory activity assay). Compared with controls and free OVAgroups, asthma-like responses are significantly reduced in the LL-OVAgroup, indicating activation of regulatory CD4⁺ T cells in ourcombination oral tolerance protocol.

Conclusion

Our data demonstrate that mucosal delivery of allergen secreting L.lactis is more potent than free allergen to induce allergen-specificimmune tolerance via the induction of antigen-specific CD4⁺ regulatory Tcells, even in the setting of established hypersensitivity.

Example E Induction of Tolerance to BLG Food Allergen Following OralAdministration of L. lactis Secreting Said Allergen Introduction

Food allergy is a disease affecting approximately 2% to 5% of thepopulation. In human beings, elevated IgE antibodies as well as thepresence of IL-4-producing, antigen-specific T lymphocytes suggest aTh2-skewed mechanism. Here, we demonstrate that oral delivery of a foodallergen by L. lactis suppresses allergen-specific immune responses viathe induction of antigen-specific CD4⁺ regulatory T cells.

Material and Methods to the Examples Bacteria and Plasmids

The L. lactis strain MG1363 is used throughout this study. Bacteria arecultured in GM17 medium, i.e. M17 (Difco Laboratories, Detroit, Mich.)supplemented with 0.5% glucose. Stock suspensions of all strains arestored at ±20° C. in 50% glycerol in GM17. For intragastricinoculations, stock suspensions are diluted 200-fold in fresh GM17 andincubated at 30° C. They reach a saturation density of 2×10⁹colony-forming units (CFU) per mL within 16 hours. Bacteria areharvested by centrifugation and concentrated 10-fold in BM9 medium. Fortreatment, each mouse receives 100 μL of this suspension daily byintragastric catheter. Bovine β-lactoglobulin cDNA is amplified andfused to the Usp45 secretion signal of the erythromycin resistant pT1 NXvector, downstream of the lactococcal P1 promotor. MG1363 strainstransformed with plasmids carrying murine BLG is designated LL-BLG.LL-pT1 NX, which is MG1363 containing the empty vector pT1 NX, serve ascontrol.

Quantification of Bovine β-Lactoglobulin (BLG)

BLG from LL-BLG is determined using an in house developed BLG-specificenzyme-linked immunosorbent assay (ELISA) and Western blot analysis.

Experimental Setting

The murine model of food allergy used to explore the protective effectof L. lactis is a mouse model of food-induced IgE-type response asdescribed by Frossard et al. (J Allergy Clin Immunol 113:958-964, 2004).Mice receive LL-BLG or an irrelevant antigen (OVA) as negative control.As a positive control for tolerance induction, mice receive a high doseof BLG in the drinking water that prevents the mice from anaphylaxisupon oral challenge with BLG.

In a prophylactic setting, the engineered L. lactis bacteria thatproduce BLG are administered orally to the mice using a gastriccatheter, using different treatment intervals and doses. Subsequently,these recipient mice are orally challenged with purified BLG antigen, inthe presence of cholera toxin. Control animals are exposed to L. lactisengineered with a control vector that does not express BLG (but OVAinstead). Induction of tolerance is assessed by analysis of anaphylaxisafter intragastric antigen challenge, by measuring BLG-specific IgG1,IgG2a and IgE titers in serum and faeces, by determining the number ofantibody secreting cells in spleen and PP, by analysis of the T cellproliferation and cytokine production in MLN, PP and spleen.

To evaluate whether the induction of immune tolerance towards BLG couldbe enhanced by L. lactis, mice are administered with LL-BLG or with 1 μgfree BLG.

Oral Sensitization to BLG.

Four- to 5-week-old female C3H/HeOuJ mice (Charles River) are immunizedat days 0, 7, 14, and 21 by intragastric gavage with 20 mg of BLG(Sigma) and 10 μg of CTX, purchased from List Biological Laboratories in0.2 mol/L NaHCO₃. The positive control group (tolerized mice) receive0.8 mg/mL BLG in their drinking water ad libitum for 4 weeks. The totalamount of protein given (22.4 mg) is similar to the total amount of BLGgiven to the sensitized mice. To demonstrate that the tolerizationprocedure also enduringly activate the peripheral and not only themucosal immune system, a group of tolerized mice is injected twice with80 μg ip BLG adsorbed to 1 mg alum at days 28 and 42.

Antigen Challenge

On day 28, all mice are challenged by intragastric gavage with 100 mgBLG in 0.4 mL 0.2 mol NaHCO3. Anaphylaxis is observed and graded byusing a reaction score (0, no reaction, to 3, severe reaction or death)described in detail elsewhere (Frosssard et al., 2001). The core bodytemperature is measured by infrared at the ear before challenge and 30minutes after gavage. The animals are killed, and blood is collected bycardiac puncture into EDTA-containing tubes, and plasma is obtained forhistamine measurement by commercial ELISA kit (Immunotech, Marseille,France).

Cell Cultures, Proliferation and Cytokine Assay

Single cell suspensions of spleen, mesenteric lymph nodes and PP areprepared as described by Frossard et al. (2004). CD4⁺ T cells andCD4⁺CD25⁻ T cells are enriched using CD4⁺ T cell isolation kit (MiltenyiBiotec, Germany) or CD4⁺CD25⁺ Regulatory T cell isolation kit (MiltenyiBiotec, Germany), respectively and MACS columns (midiMACS; MiltenyiBiotec).

Proliferation assays of bulk splenocyte and LN populations, 2×10⁵ cellsare cultured in 96-well U-bottom plates in a total volume of 200 μlcomplete medium either alone or with purified BLG, and either with orwithout anti-IL-10 or anti-TGF-β neutralising monoclonal antibodies. BLGis added at concentrations ranging from 1 to 100 μg/ml. The neutralizingantibodies are added at 1, 0.1 and 0.01 μg/ml. For proliferation assaysof CD4⁺ T cells and CD4⁺CD25⁻ T cell populations, 2×10⁵ cells CD4⁺ Tcells or CD4⁺CD25⁻ T cells are cultured in 96-well U-bottom plates withmitomycin treated splenocytes that are loaded with 1 mg/ml BLG for 16 h,acting as antigen presenting cells, at ratio's CD4⁺ T cell or CD4⁺CD25⁻T cell/APCs 1/1, 1/0.3, 1/0.1, 1/0.03, 1/0 in a total volume of 200 μlcomplete medium either with or without neutralizing antibodies. After 72h at 37° C. in a 5% CO₂ humidified incubator, proliferation is assessedby addition of 1 μCi/well [³H]-thymidin. DNA-bound radioactivity isharvested 18 h later onto glass fiber filter mats (Perkin Elmer, Boston,USA) and thymidine-incorporation is measured on a scintillation counter(Perkin Elmer).

For cytokine measurements, supernatants of the cell cultures used in thedifferent proliferation assays is collected after 24, 48 and 72 h ofculture and frozen at −80° C. until cytokine analysis will be performed.Cytokine production is quantified using the Mouse InflammationCytometric Bead Assay (BD Biosciences, Mountain View, Calif., USA).

In Vivo T Regulatory Activity Assay

In order to test for active suppression of antibody formation in mice,splenocytes, bead-purified CD4⁺ T cells, CD4⁺CD25⁻ or CD4⁺CD25⁺ T cellsisolated from the different experimental L. Lactis-treated groups areadoptively transferred to naïve C3H/HeOuJ mice. Untreated mice are usedas control. The number of transferred cells is 10⁷ for whole spleencells, subpopulation-depleted spleen cells, or positively selected CD4⁺cells and CD4⁺CD25 and CD4⁺CD25⁺ T cells. If Tregs are implicated,subsequent challenge of these mice with BLG antigen should preventinduction of humoral immune responses against BLG and anaphylaxis.

Enzyme-Linked Immunoassays for BLG-Specific Serum and Feces Antibodies.

Sera are obtained from tail bleedings at day 0, 7, 14, 21 and 28. Fecesare obtained at the same times and resuspended in PBS plus 1% FCS (Lifetechnologies) supplemented with pepstatin 1:1000 (Fluka) at 0.1 mg/mL.The samples are mechanically disaggregated and vortexed for 2 minutes,followed by two centrifugations at 4° C. for 20 minutes at 14,000 rpm.

Sera and feces are assayed for BLG-specific IgE, IgG1, IgG2a and/or IgAantibody levels by a method adapted from Adel-Patient et al. (2000, J.Immunol Methods). In brief, MaxiSorp microtiter plates (Nunc) are coatedfor 18 hours at room temperature with 250 ng/well streptavidin (Fluka),followed by 300 μL of a solution of polyvinylpyroliddon K25 (Fluka)overnight. One microgram of biotinylated BLG is incubated for 3 hours,and diluted sera (1:6666 and 1:2222 for IgG1, 1:666 and 1:222 for IgG2a,1:66 and 1:22 for IgE) or feces (1:3, 1:10, and 1:33) in PBS plus 10%horse serum is added in duplicates in presence of 0.5 μg/mL goatanti-mouse IgA, rat anti-mouse IgG1 or anti-mouse IgG2aperoxidase-labeled antibodies (Southern Biotechnologies) for 2 hours.For IgE measurement, a monoclonal rat anti-mouse IgE Ab (clone R35-72,BD Pharmingen) followed by peroxidase-coupled anti-rat Ab (Caltag) isadded. Optical density is measured at 490 nm. Results are expressed asarbitrary units, with pooled sera from BLG plus alumimmunized mice usedas a reference serum.

Antigen-Specific Antibody Production is Measured by Means of ELISPOT.

Peyer's patches are excised mechanically from the gut and incubated for30 minutes in HBSS medium supplemented with 5 mmol EDTA (LifeTechnologies). Similarly, Peyer patches and mesenteric lymph nodes aregently crushed and filtered through a 70-m nylon filter. Spleen cellsare preincubated for 5 minutes in Tris-buffered NH₄Cl to remove redblood cells. Lymphoblasts are isolate on a Percoll 60%/66% gradient(Amersham).

For the measurement of BLG-specific IgG1, IgG2a and IgA antibodies,ELISPOT plates (Millipore) are coated with streptavidin overnight at 37°C., followed by addition of 1 g of biotinylated BLG for 3 hours.Lymphoblasts isolated on a Percoll 60%166% gradient from are resuspendedat two different concentrations, 1 and 2×10⁶ in Iscove's modifiedDulbecco's medium supplemented with penicillin, streptomycin,L-glutamine, gentamicin, polymixin B, and 5% FCS for 24 hours at 37° C.,followed by overnight incubation at 4° C. with anti-IgA, anti-IgG1 andanti-IgG2a antibodies (Southern Biotechnology). Amino-ethyl-carbazole,100 μL/well, is added for 10 minutes, and the spots are automaticallycounted by using the KS ELISPOT 4.2.1 Software (Zeiss) and expressed ascell-forming units per 10⁶ cells (CFU).

LL-BLG Significantly Enhances the Tolerance-Inducing Capacity of BLG inMurine Model of Food Allergy

To study the induction of oral tolerance, mice are orally fed asdescribed above (experimental setting). Addition of LL-BLG significantlyenhances the tolerance induction towards BLG as the allergen-specificproliferative response of the splenocytes is significantly reduced inthe LL-BLG group in comparison to the control and free-BLG groups.

LL-BLG potentiates oral tolerance in association with reducedBLG-specific antibody response and lowered IL-4 cytokine production inresponse to said allergen.

To study the induction of oral tolerance, mice are orally fed asdescribed above (experimental setting). BLG-specific antibody responseand cytokine production in response to said factor is determined asdescribed above. BLG-specific antibodies levels and IL-4 aresignificantly lowered in the LL-BLG group in comparison to the controland free-BLG groups.

Results LL-BLG Enhances Oral Tolerance Via CD4+ T Cells.

To assess whether CD4 T-cells mediate the induction of oral tolerance,the allergen-specific proliferative CD4 T-cell response is studied inthe splenocytes and lymph nodes. Therefore, mice are orally fed asdescribed above (experimental setting) and the allergen-specific CD4+ Tcell proliferation is determined as described in Cell cultures,proliferation and cytokine assay. The allergen-specific CD4 T-cellresponse in the LL-BLG group is significantly reduced in comparison tothe control and free-BLG groups.

Antigen-Induced T Regulatory Cells Following LL-BLG Therapy Can TransferProtection From Allergic-Like Responses In Vivo

In order to test for active suppression of allergic-like responses inmice treated with the oral tolerance protocol, we adoptively transfersplenocytes from the different treated groups as described above (Invivo T regulatory activity assay). Compared with controls and free-BLGgroups, allergic-like responses are significantly reduced in the LL-BLGgroup, indicating activation of regulatory CD4⁺ T cells in ourcombination oral tolerance protocol.

Conclusion

Our data demonstrate that mucosal delivery of allergen secreting L.lactis is more potent than free allergen to induce allergen-specificimmune tolerance via the induction of antigen-specific CD4⁺ regulatory Tcells.

Example F Induction of Tolerance to Insulin Following OralAdministration of L. lactis Secreting Said Autoantigen Introduction

Autoimmunity is characterized by spontaneous inflammatory tissue damageand by impaired physiological function resulting from loss of toleranceto self-antigen. It is associated with a partially overactive immunesystem, which is characterized by an excess of T helper (Th) cells.Predisposing factors, such as susceptibility genes and environmentalfactors are difficult to influence, therefore recent efforts to developimmunotherapies are focused on re-establishing the functional balancebetween pathogenic effector cells and immunoregulatory T cells bydepleting the former and/or enhancing the latter. Autoimmune destructionof pancreatic islet beta cells is the major cause of Type 1 diabetesmellitus (T1D). This destruction is associated with cellular and humoralimmune responses to several beta cell autoantigens, both of which canprecede the clinical onset of disease.

Here, we demonstrate that oral delivery of an autoantigen delivering L.lactis suppresses diabetic-specific immune responses via the inductionof antigen-specific CD4⁺ regulatory T cells.

Material and Methods Bacteria and Plasmids

The L. lactis strain MG1363 is used throughout this study. Bacteria arecultured in GM17 medium, i.e. M17 (Difco Laboratories, Detroit, Mich.)supplemented with 0.5% glucose. Stock suspensions of all strains arestored at ±20° C. in 50% glycerol in GM17. For intragastricinoculations, stock suspensions are diluted 200-fold in fresh GM17 andincubated at 30° C. They reach a saturation density of 2×10⁹colony-forming units (CFU) per mL within 16 hours. Bacteria areharvested by centrifugation and concentrated 10-fold in BM9 medium. Fortreatment, each mouse receives 100 μL of this suspension daily byintragastric catheter. DNA sequence with optimal L. lactis codon usageencoding the human proinsulin II B24-C36 peptide (hpllp), porcineinsulin and immunodominant-peptide InsB₉₋₂₃ (B9-23 is essentially thesame across many species human, rat and mouse) are synthesized,amplified and fused to the Usp45 secretion signal of the erythromycinresistant pT1NX vector, downstream of the lactococcal P1 promotor.

MG1363 strains transformed with plasmids carrying murine hpllp, Insulin,InsB₉₋₂₃ are designated LL-hpllp, LL-insulin, LL-InsB₉₋₂₃. LL-pT1NX,which is MG1363 containing the empty vector pT1NX, served as control.Expression of these proteins is determined using antigen-specific ELISAand Western blot analysis.

Mice

Non-obese female and male diabetic (NOD) mice and NOD-severe combinedimmunodeficient (SCID) (Balb/c background) mice are purchased from theJackson laboratory. Balb/c wild type (WT) mice are purchased fromCharles River Italy. Mice are maintained in a specific pathogen-freecentral animal facility. Mice are treated and used in agreement with theinstitutional guidelines.

Experimental Setting

In a prophylactic setting, the LL-hpllp, LL-insulin, LL-InsB₉₋₂₃ areadministered orally to NOD mice starting from day 21 of age (weaning),and using the optimal feeding regime or until 100 days of age (when mostmice develop diabetes). In addition, LL-pT1NX is administered orally asa negative control. For the positive (tolerizing) control group,3-week-old NOD mice are treated orally with 0.8 mg humaninsulin/hpllp/InsB₉₋₂₃ for 3 times a week for 2 or 4 weeks. Developmentof diabetes is determined by continuous monitoring of urine glucoselevels three times a week and in case of glucosuria monitoring of bloodglucose levels. Pancreases are collected at 12-23 weeks and at the endof experiment (35 weeks), and serial sections are stained withhematoxylin/eosin to score mononuclear cell infiltration or byimmunohistochemistry to analyse T cell infiltration.

In a therapeutic setting the LL-hpllp, LL-insulin, LL-InsB₉₋₂₃ areadministered orally to diabetic NOD females showing stable glycosuriaand hyperglycemia (12-23 weeks). In addition, LL-pT1 NX is administeredorally as a negative control. For the positive (tolerizing) controlgroup, diabetic NOD mice are treated as described in Bresson et al.2006. Complete remission is defined as the disappearance of glycosuriaand a return to normal glycemia.

In a syngeneic islet transplantation setting, female NOD mice withrecent-onset diabetes are treated orally for 3 weeks with LL-hpllp,LL-insulin, LL-InsB₉₋₂₃, or with LL-pT1 NX as a negative control. After3 weeks, 500 freshly isolated pancreatic islets from non-diabetic NODmice are transplanted to diabetic NOD mice. Blood glucose is thenmonitored 3 times weekly until diabetes recurrence or until 15 weeksafter grafting. Animals with 2 consecutive glucose levels 250 mg/dL areconsidered diabetic and will be subsequently killed for serum collectionand histological analysis of the graft.

The precise mechanisms of tolerance induction are analyzed in vitro, invivo after re-challenging the NOD mice with specific autoantigens and byadoptive T-cell transfer into NOD-SCID mice.

Detection of Diabetes:

Glucose monitoring: urine glucose is measured by using Diastix (Miles)and is confirmed by blood glucose measurements with the blood glucosemonitoring system OneTouch Ultra (LifeScan Inc.). Diabetes is defined as2 consecutive blood glucose values superior to 250 mg/dl.

Insulitis: Mice are killed by CO₂ asphyxiation and the pancreas is fixedin 10% formalin overnight, embedded in paraffin, and serial 5 μmsections are stained with haematoxylin and eosin. The insulitis score(mean±SD) is determined by microscopically grading the degree ofcellular infiltration in 10-15 islets/mouse as follows: 0, no visiblesign of islet infiltration; 1, peri-islet infiltration; 2, <50%infiltration; 3, >50% infiltration.

Islet isolation and transplantation: Islets of insulitis- anddiabetes-free 14- to 21-day old donor NOD mice are isolated afterasceptic removal by digesting the pancreatic glands with collagenase inHanks' balanced salt solution during vigorous shaking. Islet isolationis carried out by direct hand-picking under a stereo-microscope.Diabetic recipient NOD mice were anaesthetized by intraperitonealinjection of avertin (0.02 ml/g BWT), the left kidney was exposed vialumbar incision and 500 freshly isolated islets were given under therenal capsule.

Immunohistochemistry

To detect insulin, CD4 and CD8 expression in pancreatic R cells, primaryAbs (guinea pig anti-swine insulin from Dako [dilution 1:300], anti-CD4RM4.5 and anti-CD8a IHC from BD Biosciences [dilution 1:50] are appliedto frozen tissue sections as described in Christen et al., 2004.

In Vitro Proliferation Assay

Single cell suspensions of spleen, mesenteric LN (MLNs) and PLNs areprepared. Proliferation assays of total splenocyte populations, 2×10⁵cells are cultured in 96-well U-bottom plates in a total volume of 200μl complete medium either alone or with graded concentrations (1-100μg/ml) of purified human insulin or peptides specific for CD4 T cells(InsB₉₋₂₃, H-2^(d or g) restricted) or for CD8 T cells (InsB₁₅₋₂₃, K^(d)restricted) (Sigma), and either with or without anti-IL-10 or anti-TGF-βneutralising monoclonal antibodies. The neutralizing antibodies areadded at 1, 0.1 and 0.01 μg/ml. For proliferation assays of total CD3⁺ Tcells, CD8⁺ T cells, CD4⁺ T cells and CD4⁺CD25⁻ T cell populations,0.2×10⁵ cells T cells are cultured in 96-well U-bottom plates with 1×10⁵irradiated splenocytes from WT Balb/c mice loaded with insulin or GAD65or peptides specific for CD4⁺ or CD8⁺ T cells, in a total volume of 200μl complete medium either with or without neutralizing antibodies. After72 hr at 37° C. in a 5% CO₂ humidified incubator, proliferation isassessed by addition of 1 μCi/well [³H]-thymidin. DNA-boundradioactivity is harvested 16-18 hr later onto glass fiber filter mats(Perkin Elmer, Boston, USA) and thymidine-incorporation is measured on ascintillation counter (Perkin Elmer). T-cells are purified from PLNs orspleens by negative selection through magnetic bead separation usingCD3⁺, CD4⁺ or CD8⁺ isolation kit (MACS; Milteny Biotec, Auburn, Calif.).CD4⁺ T cells are used as total cells or further separated into CD25⁺ andCD25⁻ by MACS using CD25⁺ isolation kit (Milteny Biotec). The purity(>90%) of the cell populations is determined by flow cytometricanalysis.

For cytokine measurements, supernatants of the cell cultures used in thedifferent proliferation assays (antigen-specific stimulation), describedabove, are collected after 72 h of culture and frozen at −80° C. untilcytokine analysis is performed. Cytokine production is quantified usingthe Mouse Inflammation Cytometric Bead Assay (BD Biosciences, MountainView, Calif., USA). Purified CD3⁺ T cells, CD4⁺ T or CD8⁺ T cells arecultured and stimulated in vitro non-specifically with ananti-CD3/anti-CD28 mixture (1 μg/ml each) for 24 hours or they remainunstimulated as control. The supernatants is harvested, and analysed forIL-10, IL-4, IL-5 and IFNγ production using BD™ Cytometric Bead Arrayflex set on a BD FACSArray Bioanalyzer using the FCAP array software (BDBiosciences). Capture ELISA experiments are used to determine TGF-β1using the Quantikine kit (R&D Systems).

In Vitro T Cell Proliferation Inhibition Assay

2×10⁴ purified total splenic CD4⁺CD25⁻ T cells isolated from recentlydiabetic female NOD (8-12 weeks) are co-cultured with varying numbers ofCD8⁺ T cells, CD4⁺ T cells and CD4⁺CD25⁻ T cell populations isolatedfrom the spleen, MLN or PLNs from the different experimental groups inthe presence of 2×10⁴ T-cell depleted irradiated insuline- orpetides-loaded splenocytes from WT Balb/c mice. After 72 hr at 37° C. ina 5% CO₂ humidified incubator, proliferation is assessed by addition of1 μCi/well [³H]-thymidin. DNA-bound radioactivity is harvested 16-18 hrlater onto glass fiber filter mats (Perkin Elmer, Boston, USA) andthymidine-incorporation measured on a scintillation counter (PerkinElmer).

In Vitro Cytotoxicity Assay

Lymphoblast targets used are Con A-activated splenocytes from BALB/cmice. A total of 10⁶ target cells are labelled with 100 μCi of ⁵¹Cr(Amersham International, Buckinghamshire, U.K) for 90 min at 37° C.,washed three times and then incubated with 1 μg/ml peptide (InsB₁₅₋₂₃ oran irrelevant peptide) at 37° C. for 1 h. Target cells are washed twotimes and seeded at 10⁴ cells per well. CD8⁺ T cells, isolated fromspleen, MLNs and PLNs are added to each well, in triplicate, at variouseffector:target (E:T) ratios. The plates are centrifuged at 500 rpm for2 min, and incubated at 37° C. for 4 h. After incubation, supernatantsare collected for determination of ⁵¹Cr release [% lysis=100×(testcpm−spontaneous cpm)/(total cpm−spontaneous cpm)]. For the indirectkilling assay, CD8⁺ T cells are incubated with 5 μg/ml anti-CD3 antibody(clone 145-2C11, Pharmingen) prior to incubation with effectors.

Adoptive Transfer of Diabetes

NOD-SCID mice at 8-10 wk are injected i.v. with 2×10⁷ or i.p. with 5×10⁶splenocytes isolated from diabetic female NOD mice (6 weeks, 12 weeksand 18 weeks) combined with or without graded numbers of bead-purifiedCD3⁺ T cells, CD8⁺ T cells, CD4⁺ T cells, CD4⁺CD25⁻ or CD4⁺CD25⁺ T cellsisolated from the different experimental L. Lactis-treated groups.Untreated mice are used as control. Development of diabetes isdetermined by continuous monitoring of blood glucose levels three timesa week.

Results LL-hpllp, LL-Insulin, LL-InsB₉₋₂₃ Delays Diabetes RecurrenceAfter Syngeneic Islet Transplantation

To assess whether LL-hpllp, LL-Insulin and LL-InsB(9-23) induce oraltolerance, diabetes recurrence after syngeneic islet transplantation isstudied. Therefore, mice are orally fed as described above (experimentalsetting) and pancreatic islets are transplanted as described (Isletisolation and transplantation). Diabetes recurrence is delayed in theLL-hpllp/insulin/InsB₉₋₂₃ group in comparison to the control.

LL-hpllp, LL-insulin, or LL-InsB₉₋₂₃ significantly enhances thetolerance-inducing capacity of freehpllp, insulin, or InsB₉₋₂₃ in thenon-obese diabetic mouse

To study the induction of oral tolerance, mice are orally fed asdescribed above (experimental setting). Addition of LL-hpllp,LL-insulin, LL-InsB₉₋₂₃ significantly enhances the tolerance inductiontowards autoantigen as the autoantigen-specific proliferative responseof the splenocytes is significantly reduced in theLL-hpllp/insulin/InsB₉₋₂₃ group in comparison to the control and freehpllp/insulin/InsB₉₋₂₃ groups.

LL-hpllp, LL-insulin, or LL-InsB₉₋₂₃ potentiates oral tolerance inassociation with reduced insulitis, deceased rate of beta celldestruction, and increased IL-10 production by splenocytes To study theinduction of oral tolerance, mice are orally fed as described above(experimental setting). The presence of insulitis, the rate of beta-celldestruction and cytokine production in response to said autoantigen isdetermined as described above. Histological analysis shows a significantlower degree of insulitis and beta cell destruction and increased IL-10production in the LL-hpllp/insulin/InsB₉₋₂₃ group in comparison to thecontrol and free-hpllp/insulin/InsB₉₋₂₃ groups.

LL-hpllp, LL-Insulin, LL-InsB₉₋₂₃ Enhances Oral Tolerance Via CD4+ TCells

To assess whether CD4 T cells mediate the induction of oral tolerance,the autoantigen-specific proliferative CD4 T-cell response is studied inthe splenocytes and lymph nodes. Therefore, mice are orally fed asdescribed above (experimental setting) and the autoantigen-specific CD4+T cell proliferation is determined as described (in vitro proliferationassay). The autoantigen-specific CD4 T cell response in theLL-hpllp/insulin/InsB₉₋₂₃ group in comparison to the control andfree-hpllp/insulin/InsB₉₋₂₃ groups.

Example F5 Autoaggressive CD8+ Responses are Suppressed in NOD MiceFollowing LL-InsB₉₋₂₃ Therapy

To examine whether our combination approach induce suppressive CD4+ Tcells that are capable of modulating diabetes by bystander suppressivemechanisms, we analyze the effect on CD8+ autoaggresive T cells. Thepercentage and/or activity of antigen-specific autoaggressive CD8+ cellsis strongly reduced after LL-InsB₉₋₂₃ therapy.

Antigen-Induced T Regulatory Cells Following LL-InsB₉₋₂₃ Therapy canTransfer Protection from Autoimmune-Like Responses In Vivo

In order to test for active suppression of diabetic-like responses inmice treated with the oral tolerance protocol, we adoptively transfersplenocytes from the different treated groups as described above(adoptive transfer of diabetes). Compared with controls andfree-InsB₉₋₂₃ group, diabetic-like responses are significantly reducedin the LL-InsB₉₋₂₃ group, indicating activation of regulatory CD4⁺ Tcells in our combination oral tolerance protocol.

Conclusion

We demonstrate that oral delivery of an autoantigen delivering L. lactissuppresses diabetic-specific immune responses via the induction ofantigen-specific CD4⁺ regulatory T cells.

DISCUSSION

On the whole, the above presented data indicates that oralsupplementation of a genetically modified L. lactis secreting antigenscan decrease systemic inflammation induced by that antigen, even in asensitized subject. Advantageously, the Lactococcus-mediated suppressionoften appears more potent than after mucosal administration of freeantigen. Potentially, the suppression may be mediated by the inductionof Foxp3⁺ regulatory T cells,

REFERENCES

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1. A micro-organism secreting an antigen for use in mucosal delivery totreat an immune response related disease in a patient, wherein saidimmune response related disease is chosen from the group consisting ofallergic asthma, multiple sclerosis, type I diabetes, autoimmuneuveitis, autoimmune thyroiditis, autoimmune myasthenia gravis,rheumatoid arthritis, food allergy or celiac disease.
 2. A compositionfor use in mucosal delivery to treat, prevent and/or alleviate an immuneresponse related disease chosen from the group consisting of allergicasthma, multiple sclerosis, type I diabetes, autoimmune uveitis,autoimmune thyroiditis, autoimmune myasthenia gravis, rheumatoidarthritis, food allergy or celiac disease in a patient, characterized inthat said composition comprises at least a micro-organism secreting anantigen.
 3. The composition of claim 2, wherein said micro-organism islactic acid bacterium, yeast, or Lactococcus lactis (LL).
 4. Thecomposition of claim 2, wherein said antigen is constitutively secreted.5. The composition of claim 2, wherein said micro-organism isadministered daily.
 6. The composition of claim 2, wherein said antigeninduces regulatory T-cells (Treg).
 7. The composition according to claim6, wherein said Treg cells are Foxp3+ Treg cells.
 8. The composition ofclaim 7, wherein said antigen is an immuno-dominant deamidated antigen.9. The composition of claim 8, wherein said antigen is recognized in thecontext of HLA-DQ2 or -DQ8.
 10. The composition of claim 9, wherein saidantigen is α-gliadin or hordein.
 11. The composition for use accordingto claim 10, wherein said antigen comprises or consists of SEQ ID NO: 2,4, 6 or
 8. 12. The composition of claim 2, wherein said antigen reducesproliferation of spleen and inguinal lymph node cells.
 13. Thecomposition or claim 2, wherein said antigen suppresses inflammatoryantigen specific T cell response.
 14. The composition of claim 2,wherein said mucosal delivery is selected from the group consisting ofrectal delivery, buccal delivery, pulmonary delivery, ocular delivery,nasal delivery, vaginal delivery and oral delivery.
 15. The compositionof claim 2, wherein said micro-organism is delivered for at least 1week, 1 month or 1 year.
 16. The composition of claim 2, wherein saidmicro-organism is delivered at least once or twice a day.
 17. Thecomposition of claim 2, wherein said micro-organism is delivered in adose of at least 10 femtogram to 100 mg per day.
 18. The composition ofclaim 2, wherein said micro-organism is delivered by spray, capsule,aerosol, lozenges, bolus, tablet, sachets, liquid, suspension, emulsionor troches.
 19. The composition of claim 2, wherein said composition isformulated as a medicament, medical food or nutraceutical.
 20. Methodfor treating, preventing and/or alleviating an immune response relateddisease in a patient, comprising the mucosal delivery of a compositioncomprising a micro-organism expressing an antigen, wherein said antigenis secreted, and wherein said composition is delivered daily.
 21. Themethod according to claim 20, wherein said micro-organism is lactic acidbacterium, yeast or Lactococcus lactis (LL).
 22. The method according toclaim 20, wherein disease is selected from the group consisting ofallergic asthma, multiple sclerosis, type I diabetes, autoimmuneuveitis, autoimmune thyroiditis, autoimmune myasthenia gravis,rheumatoid arthritis, food allergy and celiac disease.
 23. The methodaccording to claim 20, wherein said secretion is constitutive.
 24. Themethod according to claim 20, wherein said antigen induces regulatoryT-cells (Treg).
 25. The method according to claim 24, wherein said Tregcells are Foxp3+ Treg cells.
 26. The method according to claim 20,wherein said antigen is an immuno dominant deamidated antigen.
 27. Themethod according to claim 20, wherein said antigen is recognized in thecontext of HLA-DQ2 or -DQ8.
 28. The method according to claim 20,wherein said antigen is α-gliadin or hordein.
 29. The method accordingto claim 28, wherein said antigen comprises or consists of SEQ ID NO: 2,4, 6 or
 8. 30. The method according to claim 20, wherein said antigenreduces proliferation of spleen and inguinal lymph node cells.
 31. Themethod according to claim 20, wherein said antigen suppressesinflammatory antigen specific T cell response.
 32. The method accordingto claim 20, wherein said mucosal delivery is chosen from the groupconsisting of rectal delivery, buccal delivery, pulmonary delivery,ocular delivery, nasal delivery, vaginal delivery and oral delivery. 33.The method according to claim 20, wherein said micro-organism isdelivered for at least 1 week, 1 month or 1 year.
 34. The methodaccording to claim 20, wherein said micro-organism is delivered at leastonce or twice a day.
 35. The method according to claim 20, wherein saidmicro-organism is delivered in a dose of at least 10 femtogram to 100 mgper day.
 36. The method according to claim 20, wherein saidmicro-organism is delivered by spray, capsule, aerosol, lozenges, bolus,tablet, sachets, liquid, suspension, emulsion or troches. 37.Composition comprising a micro-organism, wherein said micro-organismconstitutively secretes an antigen, which is involved in the inductionof allergic asthma, multiple sclerosis, type I diabetes, autoimmuneuveitis, autoimmune thyroiditis, autoimmune myasthenia gravis,rheumatoid arthritis, food allergy or celiac disease.
 38. Thecomposition according to claim 37, wherein said micro-organism is lacticacid bacterium or yeast or Lactococcus lactis (LL).
 39. The compositionof claim 37, wherein said micro-organism is present in a dose of atleast 10 femtogram to 100 mg, such as 100 fg to 10 mg, or 1 pg to 1 mg,or 10 pg to 100 μg, or 100 pg to 10 μg, or 1 ng to 1 μg, or 10 ng to 100mg.
 40. Product comprising the composition of claim
 37. 41. The productof claim 40, wherein said product is selected from the group consistingof sprays, capsules, aerosols, lozenges, bolus, tablets, sachets,liquids, suspensions, emulsions and troches.
 42. Medicament,nutraceutical or medical food for treating, preventing and/oralleviating a disease or disorder involving an immune response relateddisease comprising at least a micro organism, wherein saidmicro-organism constitutively secretes an antigen, which is involved inthe induction of allergic asthma, multiple sclerosis, type I diabetes,autoimmune uveitis, autoimmune thyroiditis, autoimmune myastheniagravis, rheumatoid arthritis, food allergy or celiac disease. 43.Medicament, nutraceutical or medical food according to claim 42, whereinsaid micro organism is lactic acid bacterium or yeast or Lactococcuslactis (LL).
 44. The composition of claim 2, wherein said antigen isprepared for the continued presence in said patient.